6-amino-purin-8-one compounds

ABSTRACT

wherein R1 is C1-6alkylamino, C1-6alkoxy, or C3-7cycloalkyloxy; m is an integer having a value of 3 to 6; n is an integer having a value of 0 to 4; and salts thereof are inducers of human interferon. Compounds which induce human interferon may be useful in the treatment of various disorders, for example the treatment of allergic diseases and other inflammatory conditions for example allergic rhinitis and asthma, the treatment of infectious diseases and cancer, and may also be useful as vaccine adjuvants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/846,475 filed Dec. 19, 2017, which is a divisional of U.S. patentapplication Ser. No. 15/134,623 filed Apr. 21, 2016 which is acontinuation of U.S. patent application Ser. No. 13/272,956 filed Oct.13, 2011, now issued as U.S. Pat. No. 9,346,806 on May 24, 2016, whichis a continuation of U.S. patent application Ser. No. 12/537,392 filedAug. 7, 2009, now issued as U.S. Pat. No. 8,067,426 on Nov. 29, 2011,which claims the benefit of U.S. Provisional Application No. 61/087,777filed on Aug. 11, 2008.

BACKGROUND OF THE INVENTION

The present invention relates to compounds, processes for theirpreparation, compositions containing them, to their use in the treatmentof various disorders in particular allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma,infectious diseases, cancer, and as vaccine adjuvants.

Vertebrates are constantly threatened by the invasion of microorganismsand have evolved mechanisms of immune defence to eliminate infectivepathogens. In mammals, this immune system comprises two branches; innateimmunity and acquired immunity. The first line of host defence is theinnate immune system, which is mediated by macrophages and dendriticcells. Acquired immunity involves the elimination of pathogens at thelate stages of infection and also enables the generation ofimmunological memory. Acquired immunity is highly specific, due to thevast repertoire of lymphocytes with antigen-specific receptors that haveundergone gene rearrangement.

The innate immune response was originally thought to be non-specific,but is now known to be able to discriminate between self and a varietyof pathogens. The innate immune system recognises microbes via a limitednumber of germline-encoded Pattern-Recognition Receptors (PRRs) whichhave a number of important characteristics.

Toll-like receptors (TLRs) are a family of ten Pattern RecognitionReceptors described in man. TLRs are expressed predominantly by innateimmune cells where their role is to monitor the environment for signs ofinfection and, on activation, mobilise defence mechanisms aimed at theelimination of invading pathogens. The early innate immune-responsestriggered by TLRs limit the spread of infection, while thepro-inflammatory cytokines and chemokines that they induce lead torecruitment and activation of antigen presenting cells, B cells, and Tcells. The TLRs can modulate the nature of the adaptive immune-responsesto give appropriate protection via dendritic cell-activation andcytokine release (Akira S. et al, Nat. Immunol., 2001: 2, 675-680). Theprofile of the response seen from different TLR agonists depends on thecell type activated.

TLR7 is a member of the subgroup of TLRs (TLRs 3, 7, 8, and 9),localised in the endosomal compartment of cells which have becomespecialised to detect non-self nucleic acids. TLR7 plays a key role inanti-viral defence via the recognition of ssRNA (Diebold S. S. et al,Science, 2004: 303, 1529-1531; and Lund J. M. et al, PNAS, 2004: 101,5598-5603). TLR7 has a restricted expression-profile in man and isexpressed predominantly by B cells and plasmacytoid dendritic cells(pDC), and to a lesser extent by monocytes. Plasmacytoid DCs are aunique population of lymphoid-derived dendritic cells (0.2-0.8% ofPeripheral Blood Mononuclear Cells (PBMCs)) which are the primary type Iinterferon-producing cells secreting high levels of interferon-alpha(IFNα) and interferon-beta (IFNβ) in response to viral infections (LiuY-J, Annu. Rev. Immunol., 2005: 23, 275-306).

Allergic diseases are associated with a Th2-biased immune-response toallergens. Th2 responses are associated with raised levels of IgE,which, via its effects on mast cells, promotes a hypersensitivity toallergens, resulting in the symptoms seen, for example, in allergicrhinitis. In healthy individuals the immune-response to allergens ismore balanced with a mixed Th2/Th1 and regulatory T cell response. TLR7ligands have been shown to reduce Th2 cytokine and enhance Th1 cytokinerelease in vitro and to ameliorate Th2-type inflammatory responses inallergic lung models in vivo (Fili L. et al, J. All. Clin. Immunol.,2006: 118, 511-517; Moisan J. et al, Am. J. Physiol. Lung Cell Mol.Physiol., 2006: 290, L987-995; Tao et al, Chin. Med. J., 2006: 119,640-648). Thus TLR7 ligands have the potential to rebalance theimmune-response seen in allergic individuals and lead to diseasemodification.

Central to the generation of an effective innate immune response inmammals are mechanisms which bring about the induction of interferonsand other cytokines which act upon cells to induce a number of effects.These effects can include the activation of anti-infective geneexpression, the activation of antigen presentation in cells to drivestrong antigen-specific immunity and the promotion of phagocytosis inphagocytic cells.

Interferon was first described as a substance which could protect cellsfrom viral infection (Isaacs & Lindemann, J. Virus Interference. Proc.R. Soc. Lon. Ser. B. Biol. Sci. 1957: 147, 258-267). In man, the type Iinterferons are a family of related proteins encoded by genes onchromosome 9 and encoding at least 13 isoforms of interferon alpha(IFNα) and one isoform of interferon beta (IFNβ). Recombinant IFNα wasthe first approved biological therapeutic and has become an importanttherapy in viral infections and in cancer. As well as direct antiviralactivity on cells, interferons are known to be potent modulators of theimmune response, acting on cells of the immune system.

As a first-line therapy for hepatitis C virus (HCV) disease, interferoncombinations can be highly effective at reducing viral load and in somesubjects in eliminating viral replication. However, many patients failto show a sustained viral response and in these patients viral load isnot controlled. Additionally, therapy with injected interferon may beassociated with a number of unwanted adverse effects which are shown toaffect compliance (Dudley T, et al, Gut., 2006: 55(9), 1362-3).

Administration of a small molecule compound which could stimulate theinnate immune response, including the activation of type I interferonsand other cytokines, could become an important strategy for thetreatment or prevention of human diseases including viral infections.This type of immunomodulatory strategy has the potential to identifycompounds which may be useful not only in infectious diseases but alsoin cancer (Krieg. Curr. Oncol. Rep., 2004: 6(2), 88-95), allergicdiseases (Moisan J. et al, Am. J. Physiol. Lung Cell Mol. Physiol.,2006: 290, L987-995), other inflammatory conditions such as irritablebowel disease (Rakoff-Nahoum S., Cell., 2004, 23, 118(2): 229-41), andas vaccine adjuvants (Persing et al. Trends Microbiol. 2002: 10(10Suppl), S32-7).

In animal models, imiquimod demonstrated adjuvant activities eithertopically (Adams S. et al, J. Immunol., 2008, 181:776-84; Johnston D. etal, Vaccine, 2006, 24:1958-65), or systemically (Fransen F. et al,Infect. Immun., 2007, 75:5939-46). Resiquimod and other related TLR7/8agonists have also been shown to display adjuvant activity (Ma R. et al,Biochem. Biophys. Res. Commun., 2007, 361:537-42; Wille-Reece U. et al,Proc. Natl. Acad. Sci. USA, 2005, 102:15190-4; Wille-Reece U. et al,US2006045885 A1).

Mechanisms which lead to induction of type I interferons are only partlyunderstood. One mechanism which can lead to the induction of interferonin many cell types is the recognition of double-stranded viral RNA bythe RNA helicases RIG-I and MDA5. This mechanism is thought to be theprimary mechanism by which interferons are induced by Sendai virusinfection of cells.

Further mechanisms for the induction of interferons are viaTLR-dependent signalling events. In man, plasmacytoid dendritic cells(pDCs) are professional interferon-producing cells, able to make largeamounts of interferons in response to, for example, viral infection.These pDCs are shown to preferentially express TLR7 and TLR9 andstimulation of these receptors with viral RNA or DNA respectively caninduce expression of interferon alpha.

Oligonucleotide agonists of TLR7 and TLR9, and small moleculepurine-based agonists of TLR7 have been described which can induceinterferon alpha from these cell types in animals and in man (Takeda K.et al, Annu. Rev. Immunol., 2003: 21, 335-76). TLR7 agonists includeimidazoquinoline compounds such as imiquimod and resiquimod, oxoadenineanalogues and also nucleoside analogues such as loxoribine and7-thia-8-oxoguanosine which have long been known to induce interferonalpha. International Patent Application publication number WO2008/114008 (AstraZeneca AB/Dainippon Sumitomo Pharma Co. Ltd.)discloses 9-subsituted-8-oxoadenine compounds as TLR7 modulators.

It remains unclear how small molecule purine-like compounds can inducetype I interferons and other cytokines since the molecular targets ofthese known inducers have not been identified. However, an assaystrategy has been developed to characterise small molecule inducers ofhuman interferon IFNα (regardless of mechanism) which is based onstimulation of primary human donor cells with compounds, and isdisclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an XRPD diffractogram of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl}-7,9-dihydro-8H-purin-8-one.

FIG. 2 shows a DSC thermogram of6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl}-7,9-dihydro-8H-purin-8-one.

BRIEF DESCRIPTION OF THE INVENTION

Certain compounds of the invention have been shown to be inducers ofhuman interferon and may possess an improved profile with respect toknown inducers of human interferon, for example enhanced potency, andmay show enhanced selectivity for IFNα with respect to TNFα. Forexample, certain compounds of the invention indicate greater than1000-fold selectivity for IFNα induction over TNFα induction. Compoundswhich induce human interferon may be useful in the treatment of variousdisorders, for example the treatment of allergic diseases and otherinflammatory conditions for example allergic rhinitis and asthma, thetreatment of infectious diseases and cancer, and may also be useful asvaccine adjuvants.

Certain compounds of the invention are potent immunomodulators andaccordingly, care should be exercised in their handling.

SUMMARY OF THE INVENTION

In a first aspect, there are provided compounds of formula (I):

wherein;

R¹ is C₁₋₆alkylamino, C₁₋₆alkoxy, or C₃₋₇cycloalkyloxy;

m is an integer having a value of 3 to 6;

n is an integer having a value of 0 to 4;

with the proviso that, when m is 3 and n is 1, then R¹ is other thann-butyloxy;and salts thereof.

In a further embodiment, R¹ is C₁₋₆alkylamino or C₁₋₆alkoxy.

In a further embodiment, R¹ is n-butyloxy.

In a further embodiment, R¹ is n-butylamino.

In a further embodiment, R¹ is (1S)-1-methylbutyloxy.

In a further embodiment, R¹ is (1S)-1-methylpropyloxy.

In a further embodiment, R¹ is (1S)-1-methylpentyloxy.

In a further embodiment, R¹ is 1-methylethyloxy.

In a further embodiment, R¹ is cyclobutyloxy.

In a further embodiment, R¹ is cyclopentyloxy.

In a further embodiment, R¹ is cyclohexyloxy.

In a further embodiment, R¹ is (1R)-1-methylbutylamino.

In a further embodiment, R¹ is (1S)-1-methylbutylamino.

In a further embodiment, m is 3.

In a further embodiment, m is 4.

In a further embodiment, m is 5.

In a further embodiment, m is an integer having a value of 4 to 6.

In a further embodiment, m is 6.

In a further embodiment, n is 0.

In a further embodiment, n is 1.

In a further embodiment, n is 2.

In a further embodiment, n is 3.

In a further embodiment, n is 4.

In a further embodiment, n is an integer having a value of 2 to 4.

In a further aspect, there is provided a subset of compounds of formula(I) being compounds of formula (IA):

wherein;

R^(1A) is C₁₋₆alkylamino or C₁₋₆alkoxy;

-   -   m_(A) is an integer having a value of 3 to 6;    -   n_(A) is an integer having a value of 0 to 4;        and salts thereof.

In a further embodiment, R^(1A) is n-butyloxy.

In a further embodiment, R^(1A) is n-butylamino.

In a further embodiment, R^(1A) is (1S)-1-methylbutyloxy.

In a further embodiment, R^(1A) is (1S)-1-methylpropyloxy.

In a further embodiment, R^(1A) is (1S)-1-methylpentyloxy.

In a further embodiment, R^(1A) is 1-methylethyloxy.

In a further embodiment, R^(1A) is (1R)-1-methylbutylamino.

In a further embodiment, R^(1A) is (1S)-1-methylbutylamino.

In a further embodiment, m_(A) is 4.

In a further embodiment, m_(A) is 5.

In a further embodiment, m_(A) is 6.

In a further embodiment, n_(A) is 0.

In a further embodiment, n_(A) is 1.

In a further embodiment, n_(A) is 2.

In a further embodiment, n_(A) is 3.

In a further embodiment, n_(A) is 4.

In a further aspect, there are provided compounds of formula (IA) andsalts thereof as hereinbefore defined, wherein m is an integer having avalue of 4 to 6.

In a further aspect, there are provided compounds of formula (IA) andsalts thereof as hereinbefore defined, with the proviso that6-amino-2-(butyloxy)-9-[3-(1-pyrrolidinyl)propyl]-7,9-dihydro-8H-purin-8-oneis excluded.

In a further aspect, there are provided compounds of formula (I) andsalts thereof with the proviso that 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneand salts thereof are excluded.

In a further aspect, there are provided compounds of formula (IA) andsalts thereof as hereinbefore defined, wherein m is an integer having avalue of 4 to 6 and 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneand salts thereof are excluded.

In a further aspect, there are provided compounds of formula (IA) andsalts thereof as hereinbefore defined, with the proviso that6-amino-2-(butyloxy)-9-[3-(1-pyrrolidinyl)propyl]-7,9-dihydro-8H-purin-8-one,and6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneand salts thereof are excluded.

Examples of compounds of formula (I) are provided in the following list,and form a further aspect of the invention:

-   6-amino-9-[3-(1-azetidinyl)propyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[3-(1-pyrrolidinyl)propyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[3-(hexahydro-1H-azepin-1-yl)propyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-9-[4-(1-azetidinyl)butyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[4-(1-pyrrolidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-9-[5-(1-azetidinyl)pentyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[5-(1-pyrrolidinyl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[5-(hexahydro-1    (2H)-azocinyl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[6-(1-pyrrolidinyl)hexyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[6-(1-piperidinyl)    hexyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butyloxy)-9-[6-(hexahydro-1H-azepin-1-yl)hexyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(butylamino)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylbutyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-2-{[(1    S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-2-{[(1    S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylpropyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylpentyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-[(1-methylethyl)oxy]-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(cyclobutyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(cyclopentyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-(cyclohexyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1R)-1-methylbutyl]amino}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylbutyl]amino}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylbutyl]oxy}-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one;-   6-amino-2-{[(1    S)-1-methylpropyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,    and;-   6-amino-2-(butyloxy)-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one;    and salts thereof.

In a further embodiment, there is provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneor a salt thereof.

In a further embodiment, there is provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneor a pharmaceutically acceptable salt thereof.

In a further embodiment, there is provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneas a free base.

There is thus provided as a further aspect of the invention a compoundof formula (I), or a pharmaceutically acceptable salt thereof, for usein therapy.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for use in therapy.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for use in therapy.

It will be appreciated that, when a compound of formula (I) or apharmaceutically acceptable salt thereof is used in therapy, it is usedas an active therapeutic agent.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic diseases and other inflammatory conditions, infectiousdiseases, and cancer.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for use in the treatmentof allergic diseases and other inflammatory conditions, infectiousdiseases, and cancer.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for use in the treatment of allergic diseases and otherinflammatory conditions, infectious diseases, and cancer.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofallergic rhinitis.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for use in the treatmentof allergic rhinitis.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for use in the treatment of allergic rhinitis.

There is also therefore provided a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofasthma.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for use in the treatmentof asthma.

There is also therefore provided6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for use in the treatment of asthma.

There is also therefore provided a vaccine adjuvant comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof.

There is further provided an immugenic composition comprising an antigenor antigen composition and a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a vaccine composition comprising an antigen orantigen composition and a compound of formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, an immugenic composition comprising an antigenor antigen composition and a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treating or preventing diseasecomprising the administration to a human subject suffering from orsusceptible to disease, a vaccine composition comprising an antigen orantigen composition and a compound of formula (I), or a pharmaceuticallyacceptable salt thereof.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of animmugenic composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of avaccine composition comprising an antigen or antigen composition, forthe treatment or prevention of disease.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic diseases and other inflammatoryconditions, infectious diseases, and cancer.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic diseases and other inflammatoryconditions, infectious diseases, and cancer.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for the manufacture of a medicament for the treatment ofallergic diseases and other inflammatory conditions, infectiousdiseases, and cancer.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic rhinitis.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of allergic rhinitis.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for the manufacture of a medicament for the treatment ofallergic rhinitis.

There is further provided the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of asthma.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, for the manufacture of amedicament for the treatment of asthma.

There is further provided the use of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, for the manufacture of a medicament for the treatment ofasthma.

There is further provided a method of treatment of allergic diseases andother inflammatory conditions, infectious diseases, and cancer, whichmethod comprises administering to a human subject in need thereof, atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of allergic diseases andother inflammatory conditions, infectious diseases, and cancer, whichmethod comprises administering to a human subject in need thereof, atherapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof.

There is further provided a method of treatment of allergic diseases andother inflammatory conditions, infectious diseases, and cancer, whichmethod comprises administering to a human subject in need thereof, atherapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneas a free base.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof.

There is further provided a method of treatment of allergic rhinitis,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneas a free base.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof.

There is further provided a method of treatment of asthma, which methodcomprises administering to a human subject in need thereof, atherapeutically effective amount of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneas a free base.

The invention provides in a further aspect, a combination comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,together with at least one other therapeutically-active agent.

The invention provides in a further aspect, a combination comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, together with at leastone other therapeutically-active agent.

The invention provides in a further aspect, a combination comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, together with at least one other therapeutically-activeagent.

There is further provided a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,and one or more pharmaceutically acceptable diluents or carriers.

There is further provided a pharmaceutical composition comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable diluents or carriers.

There is further provided a pharmaceutical composition comprising6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, and one or more pharmaceutically acceptable diluents orcarriers.

There is also provided a process for preparing a pharmaceuticalcomposition which comprises admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable diluents or carriers.

There is also provided a process for preparing a pharmaceuticalcomposition which comprises admixing6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a pharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable diluents or carriers.

There is also provided a process for preparing a pharmaceuticalcomposition which comprises admixing6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,as a free base, with one or more pharmaceutically acceptable diluents orcarriers.

The compounds of formula (I) and salts thereof may be prepared by themethodology described herein, which constitutes a further aspect of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of formula (I), which process comprises the deprotection of acompound of formula (II):

wherein R¹, m, and n are as hereinbefore defined for a compound offormula (I) and R² is C₁₋₆alkyl, and thereafter, if required, carryingout one or more of the following optional steps:(i). removing any necessary protecting group;(ii). preparing a salt of the compound so-formed.

The present invention covers all combinations of embodiments and aspectsherein described.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described in terms known and appreciated bythose skilled in the art. For ease of reference certain termshereinafter are defined. The fact that certain terms are defined,however, should not be considered as indicative that defined terms areused in a manner inconsistent with the ordinary meaning or,alternatively, that any term that is undefined is indefinite or not usedwithin the ordinary and accepted meaning. Rather, all terms used hereinare believed to describe the invention such that one of ordinary skillcan appreciate the scope of the present invention. The followingdefinitions are meant to clarify, but not limit, the terms defined.

References to ‘alkyl’ include references to both straight-chain andbranched-chain aliphatic isomers of the corresponding alkyl containingup to six carbon atoms, for example up to four carbon atoms or up to twocarbon atoms. Such references to ‘alkyl’ are also applicable when analkyl group is part of another group, for example an alkylamino oralkoxy group. Examples of such alkyl groups and groups containing alkylgroups are C₁₋₆alkyl, C₁₋₆alkylamino, and C₁₋₆alkoxy.

References to ‘cycloalkyl’ refer to monocyclic alkyl groups containingbetween three and seven carbon atoms, for example four carbon atoms, orfive carbon atoms, or six carbon atoms. Such references to ‘cycloalkyl’are also applicable when a cycloalkyl group is part of another group,for example a cycloalkoxy group. Examples of such cycloalkyl groups arecyclobutyl, cyclopentyl, and cyclohexyl.

References to ‘heterocycle’ or ‘heterocyclyl’ refer to a monocyclicsaturated heterocyclic aliphatic ring containing 3-7 carbon atoms andone heteroatom, which heteroatom is nitrogen. Such heterocyclic ringsare azetidine or azetidinyl, pyrrolidine or pyrrolidinyl, piperidine orpiperidinyl, hexahydroazepine or hexahydroazepinyl, and octahydroazocineor hexahydro-(2H)-azocinyl.

References to ‘halogen’ refer to iodine, bromine, chlorine or fluorine,typically bromine, chlorine, or fluorine. References to ‘halo’ refer toiodo, bromo, chloro or fluoro, typically bromo, chloro, or fluoro.

It is to be understood that references herein to compounds of theinvention mean a compound of formula (I) as the free base, or as a salt,for example a pharmaceutically acceptable salt.

Salts of the compounds of formula (I) include pharmaceuticallyacceptable salts and salts which may not be pharmaceutically acceptablebut may be useful in the preparation of compounds of formula andpharmaceutically acceptable salts thereof. Salts may be derived fromcertain inorganic or organic acids, or certain inorganic or organicbases.

The invention includes within its scope all possible stoichiometric andnon-stoichiometric forms of the salts of the compounds of formula (I).

Examples of salts are pharmaceutically acceptable salts.Pharmaceutically acceptable salts include acid addition salts and baseaddition salts. For a review on suitable salts see Berge et al., J.Pharm. Sci., 66:1-19 (1977).

Examples of pharmaceutically acceptable acid addition salts of acompound of formula (I) include hydrobromide, hydrochloride, sulphate,p-toluenesulphonate, methanesulphonate, naphthalenesulphonate, andphenylsulphonate salts.

Salts may be formed using techniques well-known in the art, for exampleby precipitation from solution followed by filtration, or by evaporationof the solvent.

Typically, a pharmaceutically acceptable acid addition salt can beformed by reaction of a compound of formula (I) with a suitable strongacid (such as hydrobromic, hydrochloric, sulphuric, p-toluenesulphonic,methanesulphonic or naphthalenesulphonic acids), optionally in asuitable solvent such as an organic solvent, to give the salt which isusually isolated for example by crystallisation and filtration.

It will be appreciated that many organic compounds can form complexeswith solvents in which they are reacted or from which they areprecipitated or crystallised. These complexes are known as “solvates”.For example, a complex with water is known as a “hydrate”. Solvents withhigh boiling points and/or solvents with a high propensity to formhydrogen bonds such as water, ethanol, iso-propyl alcohol, and N-methylpyrrolidinone may be used to form solvates. Methods for theidentification of solvated include, but are not limited to, NMR andmicroanalysis. Solvates of the compounds of formula (I) are within thescope of the invention. As used herein, the term solvate encompassessolvates of both a free base compound as well as any salt thereof.

Certain of the compounds of the invention may contain chiral atomsand/or multiple bonds, and hence may exist in one or more stereoisomericforms. The present invention encompasses all of the stereoisomers of thecompounds of the invention, including optical isomers, whether asindividual stereoisomers or as mixtures thereof including racemicmodifications. Any stereoisomer may contain less than 10% by weight, forexample less than 5% by weight, or less than 0.5% by weight, of anyother stereoisomer. For example, any optical isomer may contain lessthan 10% by weight, for example less than 5% by weight, or less than0.5% by weight, of its antipode.

Certain of the compounds of the invention may exist in tautomeric forms.It will be understood that the present invention encompasses all of thetautomers of the compounds of the invention whether as individualtautomers or as mixtures thereof.

The compounds of the invention may be in crystalline or amorphous form.Furthermore, some of the crystalline forms of the compounds of theinvention may exist as polymorphs, all of which are included within thescope of the present invention. The most thermodynamically stablepolymorphic form or forms of the compounds of the invention are ofparticular interest.

Polymorphic forms of compounds of the invention may be characterised anddifferentiated using a number of conventional analytical techniques,including, but not limited to, X-ray powder diffraction (XRPD), infraredspectroscopy (IR), Raman spectroscopy, differential scanning calorimetry(DSC), thermogravimetric analysis (TGA) and solid-state nuclear magneticresonance (ssNMR).

It will be appreciated from the foregoing that included within the scopeof the invention are solvates, hydrates, isomers and polymorphic formsof the compounds of formula (I) and salts and solvates thereof.

Examples of disease states in which the compounds of formula (I) andpharmaceutically acceptable salts thereof have potentially beneficialeffects include allergic diseases and other inflammatory conditions forexample allergic rhinitis and asthma, infectious diseases, and cancer.The compounds of formula (I) and pharmaceutically acceptable saltsthereof are also of potential use as vaccine adjuvants.

As modulators of the immune response the compounds of formula (I) andpharmaceutically acceptable salts thereof may also be useful, asstand-alone or in combination as an adjuvant, in the treatment and/orprevention of immune-mediated disorders, including but not limited toinflammatory or allergic diseases such as asthma, allergic rhinitis andrhinoconjuctivitis, food allergy, hypersensitivity lung diseases,eosinophilic pneumonitis, delayed-type hypersensitivity disorders,atherosclerosis, pancreatitis, gastritis, colitis, osteoarthritis,psoriasis, sarcoidosis, pulmonary fibrosis, respiratory distresssyndrome, bronchiolitis, chronic obstructive pulmonary disease,sinusitis, cystic fibrosis, actinic keratosis, skin dysplasia, chronicurticaria, eczema and all types of dermatitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment and/or prevention ofreactions against respiratory infections, including but not limited toairways viral exacerbations and tonsillitis. The compounds may also beuseful in the treatment and/or prevention of autoimmune diseasesincluding but not limited to rheumatoid arthritis, psoriatic arthritis,systemic lupus erythematosus, Sjöegrens disease, ankylosing spondylitis,scleroderma, dermatomyositis, diabetes, graft rejection, includinggraft-versus-host disease, inflammatory bowel diseases including, butnot limited to, Crohn's disease and ulcerative colitis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of infectious diseasesincluding, but not limited to, those caused by hepatitis viruses (e.g.hepatitis B virus, hepatitis C virus), human immunodeficiency virus,papillomaviruses, herpesviruses, respiratory viruses (e.g. influenzaviruses, respiratory syncytial virus, rhinovirus, metapneumovirus,parainfluenzavirus, SARS), and West Nile virus. The compounds of formula(I) and pharmaceutically acceptable salts thereof may also be useful inthe treatment of microbial infections caused by, for example, bacteria,fungi, or protozoa. These include, but are not limited to, tuberculosis,bacterial pneumonia, aspergillosis, histoplasmosis, candidosis,pneumocystosis, leprosy, chlamydia, cryptococcal disease,cryptosporidosis, toxoplasmosis, leishmania, malaria, andtrypanosomiasis.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be useful in the treatment of various cancers, inparticular the treatment of cancers that are known to be responsive toimmunotherapy and including, but not limited to, renal cell carcinoma,lung cancer, breast cancer, colorectal cancer, bladder cancer, melanoma,leukaemia, lymphomas and ovarian cancer.

It will be appreciated by those skilled in the art that referencesherein to treatment or therapy may, depending on the condition, extendto prophylaxis as well as the treatment of established conditions.

As mentioned herein, compounds of formula (I) and pharmaceuticallyacceptable salts thereof may be useful as therapeutic agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be formulated for administration in any convenient way.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may, for example, be formulated for oral, topical, inhaled,intranasal, buccal, parenteral (for example intravenous, subcutaneous,intradermal, or intramuscular) or rectal administration. In one aspect,the compounds of formula (I) and pharmaceutically acceptable saltsthereof are formulated for oral administration. In a further aspect, thecompounds of formula (I) and pharmaceutically acceptable salts thereofare formulated for topical administration, for example intranasal orinhaled administration.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch, cellulose or polyvinylpyrrolidone; fillers, for example, lactose, microcrystalline cellulose,sugar, maize starch, calcium phosphate or sorbitol; lubricants, forexample, magnesium stearate, stearic acid, talc, polyethylene glycol orsilica; disintegrants, for example, potato starch, croscarmellose sodiumor sodium starch glycollate; or wetting agents such as sodium laurylsulphate. The tablets may be coated according to methods well known inthe art.

Oral liquid preparations may be in the form of, for example, aqueous oroily suspensions, solutions, emulsions, syrups or elixirs, or may bepresented as a dry product for constitution with water or other suitablevehicle before use. Such liquid preparations may contain conventionaladditives such as suspending agents, for example, sorbitol syrup, methylcellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose,carboxymethyl cellulose, aluminium stearate gel or hydrogenated ediblefats; emulsifying agents, for example, lecithin, sorbitan monooleate oracacia; non-aqueous vehicles (which may include edible oils), forexample almond oil, fractionated coconut oil, oily esters, propyleneglycol or ethyl alcohol; or preservatives, for example, methyl or propylp-hydroxybenzoates or sorbic acid. The preparations may also containbuffer salts, flavouring, colouring and/or sweetening agents (e.g.mannitol) as appropriate.

Compositions for intranasal administration include aqueous compositionsadministered to the nose by drops or by pressurised pump. Suitablecompositions contain water as the diluent or carrier for this purpose.Compositions for administration to the lung or nose may contain one ormore excipients, for example one or more suspending agents, one or morepreservatives, one or more surfactants, one or more tonicity adjustingagents, one or more co-solvents, and may include components to controlthe pH of the composition, for example a buffer system. Further, thecompositions may contain other excipients such as antioxidants, forexample sodium metabisulphite, and taste-masking agents. Compositionsmay also be administered to the nose or other regions of the respiratorytract by nebulisation.

Intranasal compositions may permit the compound(s) of formula (I) or (a)pharmaceutically acceptable salt(s) thereof to be delivered to all areasof the nasal cavities (the target tissue) and further, may permit thecompound(s) of formula (I) or (a) pharmaceutically acceptable salt(s)thereof to remain in contact with the target tissue for longer periodsof time. A suitable dosing regime for intranasal compositions would befor the patient to inhale slowly through the nose subsequent to thenasal cavity being cleared. During inhalation the composition would beadministered to one nostril while the other is manually compressed. Thisprocedure would then be repeated for the other nostril. Typically, oneor two sprays per nostril would be administered by the above procedureone, two, or three times each day, ideally once daily. Of particularinterest are intranasal compositions suitable for once-dailyadministration.

The suspending agent(s), if included, will typically be present in anamount of from 0.1 to 5% (w/w), such as from 1.5% to 2.4% (w/w), basedon the total weight of the composition. Examples of pharmaceuticallyacceptable suspending agents include, but are not limited to, Avicel®(microcrystalline cellulose and carboxymethylcellulose sodium),carboxymethylcellulose sodium, veegum, tragacanth, bentonite,methylcellulose, xanthan gum, carbopol and polyethylene glycols.

Compositions for administration to the lung or nose may contain one ormore excipients may be protected from microbial or fungal contaminationand growth by inclusion of one or more preservatives. Examples ofpharmaceutically acceptable anti-microbial agents or preservativesinclude, but are not limited to, quaternary ammonium compounds (forexample benzalkonium chloride, benzethonium chloride, cetrimide,cetylpyridinium chloride, lauralkonium chloride and myristyl picoliniumchloride), mercurial agents (for example phenylmercuric nitrate,phenylmercuric acetate and thimerosal), alcoholic agents (for examplechlorobutanol, phenylethyl alcohol and benzyl alcohol), antibacterialesters (for example esters of para-hydroxybenzoic acid), chelatingagents such as disodium edetate (EDTA) and other anti-microbial agentssuch as chlorhexidine, chlorocresol, sorbic acid and its salts (such aspotassium sorbate) and polymyxin. Examples of pharmaceuticallyacceptable anti-fungal agents or preservatives include, but are notlimited to, sodium benzoate, sorbic acid, sodium propionate,methylparaben, ethylparaben, propylparaben and butylparaben. Thepreservative(s), if included, may be present in an amount of from 0.001to 1% (w/w), such as from 0.015% to 0.5% (w/w) based on the total weightof the composition.

Compositions (for example wherein at least one compound is insuspension) may include one or more surfactants which functions tofacilitate dissolution of the medicament particles in the aqueous phaseof the composition. For example, the amount of surfactant used is anamount which will not cause foaming during mixing. Examples ofpharmaceutically acceptable surfactants include fatty alcohols, estersand ethers, such as polyoxyethylene (20) sorbitan monooleate(Polysorbate 80), macrogol ethers, and poloxamers. The surfactant may bepresent in an amount of between about 0.01 to 10% (w/w), such as from0.01 to 0.75% (w/w), for example about 0.5% (w/w), based on the totalweight of the composition.

One or more tonicity-adjusting agent(s) may be included to achievetonicity with body fluids e.g. fluids of the nasal cavity, resulting inreduced levels of irritancy. Examples of pharmaceutically acceptabletonicity-adjusting agents include, but are not limited to, sodiumchloride, dextrose, xylitol, calcium chloride, glucose, glycerine andsorbitol. A tonicity-adjusting agent, if present, may be included in anamount of from 0.1 to 10% (w/w), such as from 4.5 to 5.5% (w/w), forexample about 5.0% (w/w), based on the total weight of the composition.

The compositions of the invention may be buffered by the addition ofsuitable buffering agents such as sodium citrate, citric acid,trometamol, phosphates such as disodium phosphate (for example thedodecahydrate, heptahydrate, dihydrate and anhydrous forms), or sodiumphosphate and mixtures thereof.

A buffering agent, if present, may be included in an amount of from 0.1to 5% (w/w), for example 1 to 3% (w/w) based on the total weight of thecomposition.

Examples of taste-masking agents include sucralose, sucrose, saccharinor a salt thereof, fructose, dextrose, glycerol, corn syrup, aspartame,acesulfame-K, xylitol, sorbitol, erythritol, ammonium glycyrrhizinate,thaumatin, neotame, mannitol, menthol, eucalyptus oil, camphor, anatural flavouring agent, an artificial flavouring agent, andcombinations thereof.

One or more co-solvent(s) may be included to aid solubility of themedicament compound(s) and/or other excipients. Examples ofpharmaceutically acceptable co-solvents include, but are not limited to,propylene glycol, dipropylene glycol, ethylene glycol, glycerol,ethanol, polyethylene glycols (for example PEG300 or PEG400), andmethanol. In one embodiment, the co-solvent is propylene glycol.

Co-solvent(s), if present, may be included in an amount of from 0.05 to30% (w/w), such as from 1 to 25% (w/w), for example from 1 to 10% (w/w)based on the total weight of the composition.

Compositions for inhaled administration include aqueous, organic oraqueous/organic mixtures, dry powder or crystalline compositionsadministered to the respiratory tract by pressurised pump or inhaler,for example, reservoir dry powder inhalers, unit-dose dry powderinhalers, pre-metered multi-dose dry powder inhalers, nasal inhalers orpressurised aerosol inhalers, nebulisers or insufflators. Suitablecompositions contain water as the diluent or carrier for this purposeand may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous compositions mayalso be administered to the nose and other regions of the respiratorytract by nebulisation. Such compositions may be aqueous solutions orsuspensions or aerosols delivered from pressurised packs, such as ametered dose inhaler, with the use of a suitable liquefied propellant.

Compositions for administration topically to the nose (for example, forthe treatment of rhinitis) or to the lung, include pressurised aerosolcompositions and aqueous compositions delivered to the nasal cavities bypressurised pump. Compositions which are non-pressurised and aresuitable for administration topically to the nasal cavity are ofparticular interest. Suitable compositions contain water as the diluentor carrier for this purpose. Aqueous compositions for administration tothe lung or nose may be provided with conventional excipients such asbuffering agents, tonicity-modifying agents and the like. Aqueouscompositions may also be administered to the nose by nebulisation.

A fluid dispenser may typically be used to deliver a fluid compositionto the nasal cavities. The fluid composition may be aqueous ornon-aqueous, but typically aqueous. Such a fluid dispenser may have adispensing nozzle or dispensing orifice through which a metered dose ofthe fluid composition is dispensed upon the application of auser-applied force to a pump mechanism of the fluid dispenser. Suchfluid dispensers are generally provided with a reservoir of multiplemetered doses of the fluid composition, the doses being dispensable uponsequential pump actuations. The dispensing nozzle or orifice may beconfigured for insertion into the nostrils of the user for spraydispensing of the fluid composition into the nasal cavity. A fluiddispenser of the aforementioned type is described and illustrated inInternational Patent Application publication number WO 2005/044354(Glaxo Group Limited). The dispenser has a housing which houses afluid-discharge device having a compression pump mounted on a containerfor containing a fluid composition. The housing has at least onefinger-operable side lever which is movable inwardly with respect to thehousing to move the container upwardly in the housing by means of a camto cause the pump to compress and pump a metered dose of the compositionout of a pump stem through a nasal nozzle of the housing. In oneembodiment, the fluid dispenser is of the general type illustrated inFIGS. 30-40 of WO 2005/044354.

Aqueous compositions containing a compound of formula (I) or apharmaceutically acceptable salt thereof may also be delivered by a pumpas disclosed in International Patent Application publication numberWO2007/138084 (Glaxo Group Limited), for example as disclosed withreference to FIGS. 22-46 thereof, or as disclosed in United Kingdompatent application number GB0723418.0 (Glaxo Group Limited), for exampleas disclosed with reference to FIGS. 7-32 thereof. The pump may beactuated by an actuator as disclosed in FIGS. 1-6 of GB0723418.0.

Dry powder compositions for topical delivery to the lung by inhalationmay, for example, be presented in capsules and cartridges of for examplegelatine, or blisters of for example laminated aluminium foil, for usein an inhaler or insufflator. Powder blend compositions generallycontain a powder mix for inhalation of the compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable powder base(carrier/diluent/excipient substance) such as mono-, di-, orpolysaccharides (for example lactose or starch). Dry powder compositionsmay also include, in addition to the drug and carrier, a furtherexcipient (for example a ternary agent such as a sugar ester for examplecellobiose octaacetate, calcium stearate, or magnesium stearate.

In one embodiment, a composition suitable for inhaled administration maybe incorporated into a plurality of sealed dose containers provided onmedicament pack(s) mounted inside a suitable inhalation device. Thecontainers may be rupturable, peelable, or otherwise openableone-at-a-time and the doses of the dry powder composition administeredby inhalation on a mouthpiece of the inhalation device, as known in theart. The medicament pack may take a number of different forms, forinstance a disk-shape or an elongate strip. Representative inhalationdevices are the DISKHALER™ and DISKUS™ devices, marketed byGlaxoSmithKline.

A dry powder inhalable composition may also be provided as a bulkreservoir in an inhalation device, the device then being provided with ametering mechanism for metering a dose of the composition from thereservoir to an inhalation channel where the metered dose is able to beinhaled by a patient inhaling at a mouthpiece of the device. Exemplarymarketed devices of this type are TURBUHALER™ (AstraZeneca), TWISTHALER™(Schering) and CLICKHALER™ (Innovata.)

A further delivery method for a dry powder inhalable composition is formetered doses of the composition to be provided in capsules (one doseper capsule) which are then loaded into an inhalation device, typicallyby the patient on demand. The device has means to rupture, pierce orotherwise open the capsule so that the dose is able to be entrained intothe patient's lung when they inhale at the device mouthpiece. Asmarketed examples of such devices there may be mentioned ROTAHALER™(GlaxoSmithKline) and HANDIHALER™ (Boehringer Ingelheim.)

Pressurised aerosol compositions suitable for inhalation can be either asuspension or a solution and may contain a compound of formula (I) or apharmaceutically acceptable salt thereof and a suitable propellant suchas a fluorocarbon or hydrogen-containing chlorofluorocarbon or mixturesthereof, particularly hydrofluoroalkanes, especially1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane or amixture thereof. The aerosol composition may optionally containadditional composition excipients well known in the art such assurfactants e.g. oleic acid, lecithin or an oligolactic acid orderivative thereof e.g. as described in WO 94/21229 and WO 98/34596(Minnesota Mining and Manufacturing Company) and co-solvents e.g.ethanol. Pressurised compositions will generally be retained in acanister (e.g. an aluminium canister) closed with a valve (e.g. ametering valve) and fitted into an actuator provided with a mouthpiece.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, wool-fat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may, for example, be formulated for transdermal delivery bycomposition into patches or other devices (e.g. pressurised gas devices)which deliver the active component into the skin.

For buccal administration the compositions may take the form of tabletsor lozenges formulated in the conventional manner.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated as suppositories, e.g. containingconventional suppository bases such as cocoa butter or other glycerides.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated for parenteral administration by bolusinjection or continuous infusion and may be presented in unit dose form,for instance as ampoules, vials, small volume infusions or pre-filledsyringes, or in multidose containers with an added preservative. Thecompositions may take such forms as solutions, suspensions, or emulsionsin aqueous or non-aqueous vehicles, and may contain formulatory agentssuch as anti-oxidants, buffers, antimicrobial agents and/or tonicityadjusting agents. Alternatively, the active ingredient may be in powderform for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use. The dry solid presentation may beprepared by filling a sterile powder aseptically into individual sterilecontainers or by filling a sterile solution aseptically into eachcontainer and freeze-drying.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may also be formulated with vaccines as adjuvants to modulatetheir activity. Such compositions may contain antibody(ies) or antibodyfragment(s) or an antigenic component including but not limited toprotein, DNA, live or dead bacteria and/or viruses or virus-likeparticles, together with one or more components with adjuvant activityincluding but not limited to aluminium salts, oil and water emulsions,heat shock proteins, lipid A preparations and derivatives, glycolipids,other TLR agonists such as CpG DNA or similar agents, cytokines such asGM-CSF or IL-12 or similar agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be employed alone or in combination with other therapeuticagents. The compounds of formula (I) and pharmaceutically acceptablesalts thereof and the other pharmaceutically active agent(s) may beadministered together or separately and, when administered separately,administration may occur simultaneously or sequentially, in any order.The amounts of the compound(s) of formula (I) or (a) pharmaceuticallyacceptable salt(s) thereof and the other pharmaceutically activeagent(s) and the relative timings of administration will be selected inorder to achieve the desired combined therapeutic effect. Theadministration of a combination of a compound of formula (I) or apharmaceutically acceptable salt thereof with other treatment agents maybe by administration concomitantly in a unitary pharmaceuticalcomposition including both compounds, or in separate pharmaceuticalcompositions each including one of the compounds. Alternatively, thecombination may be administered separately in a sequential mannerwherein one treatment agent is administered first and the other secondor vice versa. Such sequential administration may be close in time orremote in time.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more agents useful in theprevention or treatment of viral infections. Examples of such agentsinclude, without limitation; polymerase inhibitors such as thosedisclosed in WO 2004/037818-A1, as well as those disclosed in WO2004/037818 and WO 2006/045613; JTK-003, JTK-019, NM-283, HCV-796,R-803, R1728, R1626, as well as those disclosed in WO 2006/018725, WO2004/074270, WO 2003/095441, US2005/0176701, WO 2006/020082, WO2005/080388, WO 2004/064925, WO 2004/065367, WO 2003/007945, WO02/04425, WO 2005/014543, WO 2003/000254, EP 1065213, WO 01/47883, WO2002/057287, WO 2002/057245 and similar agents; replication inhibitorssuch as acyclovir, famciclovir, ganciclovir, cidofovir, lamivudine andsimilar agents; protease inhibitors such as the HIV protease inhibitorssaquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir,brecanavir, atazanavir, tipranavir, palinavir, lasinavir, and the HCVprotease inhibitors BILN2061, VX-950, SCH503034; and similar agents;nucleoside and nucleotide reverse transcriptase inhibitors such aszidovudine, didanosine, lamivudine, zalcitabine, abacavir, stavidine,adefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine,alovudine, amdoxovir, elvucitabine, and similar agents; non-nucleosidereverse transcriptase inhibitors (including an agent havinganti-oxidation activity such as immunocal, oltipraz etc.) such asnevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz,capravirine, TMC-278, TMC-125, etravirine, and similar agents; entryinhibitors such as enfuvirtide (T-20), T-1249, PRO-542, PRO-140,TNX-355, BMS-806, 5-Helix and similar agents; integrase inhibitors suchas L-870,180 and similar agents; budding inhibitors such as PA-344 andPA-457, and similar agents; chemokine receptor inhibitors such asvicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK-427,857), TAK449, aswell as those disclosed in WO 02/74769, WO 2004/054974, WO 2004/055012,WO 2004/055010, WO 2004/055016, WO 2004/055011, and WO 2004/054581, andsimilar agents; neuraminidase inhibitors such as CS-8958, zanamivir,oseltamivir, peramivir and similar agents; ion channel blockers such asamantadine or rimantadine and similar agents; and interfering RNA andantisense oligonucleotides and such as ISIS-14803 and similar agents;antiviral agents of undetermined mechanism of action, for example thosedisclosed in WO 2005/105761, WO 2003/085375, WO 2006/122011, ribavirin,and similar agents. The compounds of formula (I) and pharmaceuticallyacceptable salts thereof may also be used in combination with one ormore other agents which may be useful in the prevention or treatment ofviral infections for example immune therapies (e.g. interferon or othercytokines/chemokines, cytokine/chemokine receptor modulators, cytokineagonists or antagonists and similar agents); and therapeutic vaccines,antifibrotic agents, anti-inflammatory agents such as corticosteroids orNSAIDs (non-steroidal anti-inflammatory agents) and similar agents.

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of allergic disease,inflammatory disease, autoimmune disease, for example; antigenimmunotherapy, anti-histamines, steroids, NSAIDs, bronchodilators (e.g.beta 2 agonists, adrenergic agonists, anticholinergic agents,theophylline), methotrexate, leukotriene modulators and similar agents;monoclonal antibody therapy such as anti-IgE, anti-TNF, anti-IL-5,anti-IL-6, anti-IL-12, anti-IL-1 and similar agents; receptor therapiese.g. entanercept and similar agents; antigen non-specificimmunotherapies (e.g. interferon or other cytokines/chemokines,cytokine/chemokine receptor modulators, cytokine agonists orantagonists, TLR agonists and similar agents).

The compounds of formula (I) and pharmaceutically acceptable saltsthereof may be used in combination with one or more other agents whichmay be useful in the prevention or treatment of cancer, for examplechemotherapeutics such as alkylating agents, topoisomerase inhibitors,antimetabolites, antimitotic agents, kinase inhibitors and similaragents; monoclonal antibody therapy such as trastuzumab, gemtuzumab andother similar agents; and hormone therapy such as tamoxifen, goserelinand similar agents.

The pharmaceutical compositions according to the invention may also beused alone or in combination with at least one other therapeutic agentin other therapeutic areas, for example gastrointestinal disease. Thecompositions according to the invention may also be used in combinationwith gene replacement therapy.

The invention includes in a further aspect a combination comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,together with at least one other therapeutically active agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical composition and thus pharmaceuticalcompositions comprising a combination as defined above together with atleast one pharmaceutically acceptable diluent or carrier thereofrepresent a further aspect of the invention.

A therapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof will depend upon a number offactors. For example, the species, age, and weight of the recipient, theprecise condition requiring treatment and its severity, the nature ofthe composition, and the route of administration are all factors to beconsidered. The therapeutically effective amount ultimately should be atthe discretion of the attendant physician. Regardless, an effectiveamount of a compound of the present invention for the treatment ofhumans suffering from frailty, generally, should be in the range of0.0001 to 100 mg/kg body weight of recipient per day. More usually theeffective amount should be in the range of 0.001 to 10 mg/kg body weightper day. Thus, for a 70 kg adult one example of an actual amount per daywould usually be from 7 to 700 mg. For intranasal and inhaled routes ofadministration, typical doses for a 70 kg adult should be in the rangeof 1 microgramme to 1 mg per day. This amount may be given in a singledose per day or in a number (such as two, three, four, five, or more) ofsub-doses per day such that the total daily dose is the same. Aneffective amount of a pharmaceutically acceptable salt of a compound offormula (I) may be determined as a proportion of the effective amount ofthe compound of formula (I) or a pharmaceutically acceptable saltthereof per se. Similar dosages should be appropriate for treatment ofthe other conditions referred to herein.

Compounds of formula (I) and pharmaceutically acceptable salts thereofmay also be administered at any appropriate frequency e.g. 1-7 times perweek. The precise dosing regimen will of course depend on factors suchas the therapeutic indication, the age and condition of the patient, andthe particular route of administration chosen.

Pharmaceutical compositions may be presented in unit-dose formscontaining a predetermined amount of active ingredient per unit dose.Such a unit may contain, as a non-limiting example, 0.5 mg to 1 g of acompound of formula (I) or a pharmaceutically acceptable salt thereof,depending on the condition being treated, the route of administration,and the age, weight, and condition of the patient. Preferred unit-dosagecompositions are those containing a daily dose or sub-dose, as hereinabove recited, or an appropriate fraction thereof, of an activeingredient. Such pharmaceutical compositions may be prepared by any ofthe methods well-known in the pharmacy art.

There is thus further provided a pharmaceutical composition comprising acompound of formula (I), or a pharmaceutically acceptable salt thereof,and one or more pharmaceutically acceptable diluents or carriers.

There is also provided a process for preparing such a pharmaceuticalcomposition which comprises admixing a compound of formula (I), or apharmaceutically acceptable salt thereof, with one or morepharmaceutically acceptable diluents or carriers.

Throughout the description and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

The compounds of formula (I) and salts thereof may be prepared by themethodology described hereinafter, constituting further aspects of thisinvention.

Accordingly, there is provided a process for the preparation of acompound of formula (I), which process comprises the deprotection of acompound of formula (II):

wherein R¹, m, and n are as hereinbefore defined for a compound offormula (I) and R² is C₁₋₆alkyl, and thereafter, if required, carryingout one or more of the following optional steps:(i). removing any necessary protecting group;(ii). preparing a salt of the compound so-formed.

For example, a compound of formula (II) is dissolved in a suitablesolvent in the presence of a solution of a suitable acid, for example asolution of hydrogen chloride in 1,4-dioxane and stirred at a suitabletemperature, for example ambient temperature for a suitable period oftime, for example 12-24 hours. The solvent is removed under reducedpressure and the residue is dissolved in a suitable solvent, for examplemethanol, and loaded onto an ion-exchange cartridge, for example anaminopropyl SPE cartridge. The cartridge is eluted with a suitablesolvent, for example methanol and the solvent removed to give a compoundof formula (I).

A compound of formula (II) may be prepared by reaction of a compound offormula (III):

wherein R¹ and m are as hereinbefore defined for a compound of formula(I), R² is as hereinbefore defined for a compound of formula (II), and Xis a leaving group, for example a halo group such as bromo or chloro,with a compound of formula (IV):

wherein n is as defined for a compound of formula (I).

For example, a compound of formula (III), a compound of formula (IV) anda suitable base, for example N,N-diisopropylethylamine, are dissolved ina suitable solvent, for example DMF, and heated at a suitabletemperature, for example 50-60° C. for a suitable period of time, forexample 46-50 hours. If necessary additional compound of formula (IV)and base are added and the reaction mixture heated at a suitabletemperature, for example 50-60° C. for a suitable period of time, forexample 46-50 hours. The product is then extracted from the reactionusing conventional means, for example by partitioning between a suitableorganic solvent and water, followed by isolation of the organic phaseand removal of the solvent, and purification if required.

A compound of formula (III) may be prepared by reaction of a compound offormula (V), for example a salt of a compound of formula (V) such as thetrifluoroacetate salt:

wherein R¹ is as hereinbefore defined for a compound of formula (I) andR² is as hereinbefore defined for a compound of formula (II), with acompound of formula (VI):

wherein m is as hereinbefore defined for a compound of formula (I) and Xis as hereinbefore defined for a compound of formula (III).

For example, the trifluoroacetate salt of a compound of formula (V) anda suitable base, for example potassium carbonate, are suspended in asuitable solvent, for example DMF, and heated to a suitable temperature,for example 50-60° C., under a suitable atmosphere, for example anatmosphere of nitrogen, for a suitable period of time, for example20-120 minutes. The mixture is cooled to a suitable temperature, forexample ambient temperature, and a compound of formula (VI) added andstirring continued at ambient temperature for a suitable period of time,for example 18-24 hours. The solvent is evaporated under reducedpressure and the residue partitioned between a suitable solvent, forexample DCM, and water. The crude product is then isolated from theorganic phase and purified by conventional techniques such as columnchromatography.

Alternatively, a compound of formula (II) may be prepared by reaction ofa compound of formula (V), for example a salt of a compound of formula(V) such as the trifluoroacetate salt, a compound of formula (VI)wherein X is bromo, and a compound of formula (IV) as a ‘one-pot’process.

For example, the trifluoroacetate salt of a compound of formula (V) isdissolved in a suitable solvent, for example DMF and a suitable base,for example potassium carbonate, added. The reaction mixture is stirredat a suitable temperature, for example 45-60° C. under a suitableatmosphere, for example an atmosphere of nitrogen, for a suitable periodof time, for example 1-2 hours and then cooled to a suitabletemperature, for example ambient temperature. A compound of formula (VI)wherein X is bromo is then added and, after stirring for a suitableperiod of time, for example 40-60 minutes, a compound of formula (IV)and a suitable base, for example triethylamine, in a suitable solvent,for example DMF are added. The reaction mixture is then stirred for asuitable period of time, for example 12-24 hours. The solvent is removedand the residue is partitioned between a suitable organic solvent, forexample dichloromethane, and water. The crude product of formula (II) isisolated by conventional means and purified by, for example,chromatography.

A salt of a compound of formula (V) may be prepared by deprotection of acompound of formula (VII):

wherein R¹ is as hereinbefore defined for a compound of formula (I), R²is as hereinbefore defined for a compound of formula (II), and P is aprotecting group, for example a tetrahydro-2H-pyran-2-yl group, in thepresence of a suitable acid, for example trifluoroacetic acid.

For example, a suitable acid, for example trifluoroacetic acid, is addedto a solution of a compound of formula (VII) in a suitable solvent, forexample methanol. The mixture is stirred at a suitable temperature, forexample ambient temperature, for a suitable period of time, for example48-72 hours. The reaction mixture is then concentrated under reducedpressure before being diluted with a suitable solvent, for example ethylacetate. The resultant mixture is filtered and washed with a smallvolume of a suitable solvent, for example ethyl acetate until thefiltrate is colourless. The residue is dried in air and then underreduced pressure to give the salt of a compound of formula (V). Thefiltrate may be concentrated and the concentrate diluted with a smallvolume of a suitable solvent, for example ethyl acetate, and thenfiltered and dried to yield a second crop of the salt of a compound offormula (V).

A salt of a compound of formula (V), for example the trifluoroacetatesalt, may also be prepared by reaction of a compound of formula (IX):

wherein R¹ is as hereinbefore defined for a compound of formula (I) andP is as hereinbefore defined for a compound of formula (VII), with asuitable halogenating agent, for example N-bromosuccinimide, followed byreaction with an alkoxide anion, for example a methoxide anion, and thenisolated in the presence of a suitable acid, for example trifluoroaceticacid.

For example, to a solution of crude compound of formula (IX) in asuitable dry solvent, for example dry chloroform, at a suitabletemperature, for example ambient temperature, is added a suitablehalogenating agent, for example N-bromosuccinimide, in portions over asuitable period of time, for example 5 minutes. The solution is stirredat a suitable temperature, for example ambient temperature, for asuitable period of time, for example 25-35 minutes. The reaction mixtureis then washed with water and the organic phase dried by, for example,passing through a hydrophobic frit and concentrated under reducedpressure. The resultant solid is dissolved in a suitable dry solvent,for example dry methanol, and a suitable alkoxide, for example asolution of sodium methoxide in methanol, is added at a suitabletemperature, for example ambient temperature, under an inert atmosphere,for example an atmosphere of nitrogen. The reaction mixture is heated ata suitable temperature, for example 60-70′C, with a condenser attached,for a suitable period of time, for example 12-18 hours. The reactionmixture is then cooled and concentrated under reduced pressure. Theresidue is then taken up in a suitable solvent, for example ethylacetate, and poured into a suitable aqueous medium, for examplesaturated aqueous ammonium chloride solution. The organic layer isseparated and washed further with water, dried, for example overmagnesium sulphate, filtered and concentrated under reduced pressure. Toa solution of this material in a suitable dry solvent, such as drymethanol, at a suitable temperature, for example ambient temperature, isadded a suitable acid, for example trifluoroacetic acid. The reaction isstirred for a suitable period of time, for example 25-35 hours, andconcentrated under reduced pressure to give a compound of formula (V).

A compound of formula (VII) may be prepared by reaction of a compound offormula (VIII):

wherein R¹ is as hereinbefore defined for a compound of formula (I), Pis as hereinbefore defined for a compound of formula (VII), and Q is ahalogen atom, for example a bromine atom, with an alkoxide anion, forexample methoxide anion.

For example, a solution of a compound of formula (VIII) in a suitablesolvent, for example methanol, is heated to reflux with a solution of asuitable alkoxide, for example sodium methoxide, in a suitable solvent,for example methanol, for a suitable period of time, for example 4-5hours. The reaction mixture is concentrated under reduced pressure andpartitioned between a suitable organic solvent, for example ethylacetate, and a suitable aqueous medium, for example saturated aqueousammonium chloride solution. The organic phase is separated, washed, forexample with brine, and dried by, for example passing through ahydrophobic frit. The solvent is then removed under reduced pressure togive a compound of formula (VII).

A compound of formula (VIII) may be prepared by reaction of a compoundof formula (IX) with a suitable halogenating agent, such asN-bromosuccinimide.

For example, a compound of formula (IX) is dissolved in a suitablesolvent, for example chloroform, and cooled to a suitable temperature,for example 0-0.5° C. To this solution is added a suitable halogenatingagent, such as N-bromosuccinimide, while maintaining the temperaturebelow about 3° C. The solution is stirred at a suitable temperature, forexample 2-3° C. for a suitable period of time, for example 30-45 minutesthen allowed to warm to a suitable temperature, for example ambienttemperature, and stirred for a suitable period of time, for example 5-7hours. The reaction mixture is then washed with water and the organicphase dried and separated from the aqueous phase using, for example, ahydrophobic frit. The organic solvent is then removed and the crudeproduct purified by, for example, chromatography, to give a compound offormula (VIII).

A compound of formula (IX) wherein R¹ is C₁₋₆alkoxy may be prepared byreaction of a compound of formula (X):

wherein P is as hereinbefore defined for a compound of formula (VII),and T is a suitable leaving group, for example a halogen atom, forexample a chlorine atom, or a fluorine atom, with a solution of acompound of formula (XIII):

R¹-M  (XIII)

wherein R¹ is C₁₋₆alkoxy and M is a suitable alkali metal ligand such assodium, prepared in a solvent of formula (IIIS):

R¹—H  (XIIIS)

wherein the R¹ group in the compound of formula (XIII) is the same asthe R¹ group in the solvent of formula (XIIIS).

For example, a compound of formula (XIII) such as sodium t-butoxide, isadded to a solvent of formula (XIIIS). The mixture is stirred untilhomogeneous, then a compound of formula (VII) is added. The reactionmixture is heated to a suitable temperature, for example 100C, for asuitable period of time, for example 12-18 hours. The solvent issubstantially removed under reduced pressure and partitioned between asuitable solvent, for example diethyl ether, and water. The organicphase is separated and the aqueous phase re-extracted with furthersolvent. The organic layers are then isolated, combined, dried using asuitable drying agent, for example anhydrous magnesium sulphate. Thedrying agent is removed by filtration and the solvent removed from theproduct under reduced pressure to give a compound of formula (IX)wherein R¹ is C₁₋₆alkoxy.

A compound of formula (IX) wherein R¹ is C₁₋₆alkylamino may be preparedby reaction of a compound of formula (X) with a compound of formula(XIV):

R¹—H  (XIV)

wherein R¹ is C₁₋₆alkylamino.

For example, a compound of formula (XIV) is added to a solution of acompound of formula (X) in a suitable dry solvent, for example dryethylene glycol, at a suitable temperature, for example ambienttemperature, under a suitable inert atmosphere, for example anatmosphere of nitrogen. The reaction mixture is heated at a suitabletemperature, for example 110-130° C., for a suitable period of time, forexample 12-18 hours. The reaction is then cooled to a suitabletemperature, for example ambient temperature, diluted with a suitablesolvent, for example ethyl acetate, and washed with water. The organiclayer is dried with a suitable drying agent, for example anhydrousmagnesium sulphate, filtered and concentrated under reduced pressure toyield a compound of formula (IX) wherein R¹ is C₁₋₆alkylamino.

A compound of formula (X) may be prepared by reaction of a compound offormula (XI):

wherein P is as hereinbefore defined for a compound of formula (VII),and T is as hereinbefore defined for a compound of formula (X), and V isa suitable leaving group, for example a halogen atom, for example achlorine atom, with an alcoholic solution of ammonia, for example asolution of ammonia in iso-propyl alcohol.

For example, a compound of formula (XI) is heated with an alcoholicsolution of ammonia, for example a 2M solution of ammonia in iso-propylalcohol, at a suitable temperature, for example 50-60° C., for asuitable period of time, for example 5-6 hours. The reaction mixture isthen left to stand at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 12-18 hours. Afurther quantity of the alcoholic solution of ammonia, for example a 2Msolution of ammonia in iso-propyl alcohol, is added to break up theresultant cake and the reaction mixture heated for a further period oftime, for example 8-10 hours, until the reaction is complete. Water isadded to the reaction mixture and the solid removed by filtration,washed with a suitable washing medium, for example a mixture ofiso-propyl alcohol and water, and then dried, for example by air-dryingunder suction to give a first crop of a compound of formula (X). Thefiltrate is allowed to stand for a further period of time, for example12-18 hours and the resultant second crop of a compound of formula (X)isolated by filtration and dried.

A compound of formula (X) may also be prepared by reaction of a compoundof formula (XII):

wherein T is as hereinbefore defined for a compound of formula (X), andV is as hereinbefore defined for a compound of formula (XI), with acompound of formula (XV):

P^(U)—H  (XV)

wherein P^(U) is a suitable precursor to the protecting group P, forexample a 3,4-dihydro-2H-pyranyl group, followed by reaction with analcoholic solution of ammonia, for example a solution of ammonia iniso-propyl alcohol.

For example, p-toluenesulfonic acid monohydrate is added to a solutionof a compound of formula (XII) in a suitable dry solvent, for exampledry ethyl acetate. The reaction mixture is heated to a suitabletemperature, for example 50-60° C., and a compound of formula (XV)added. The reaction is stirred at a suitable temperature, for example50-60° C., for a suitable period of time, for example 1-2 hours, and thesolvent removed under reduced pressure. A suspension of the resultantsolid in an alcoholic solution of ammonia, for example a 2M solution ofammonia in iso-propyl alcohol is heated under a suitable inertatmosphere, for example an atmosphere of nitrogen, at a suitabletemperature, for example 60-70′C, for a suitable period of time, forexample 4-5 hours with an attached condenser. The reaction mixture ispoured into water and allowed to cool for a suitable period of time, forexample 12-18 hours. The resultant precipitate is isolated by filtrationand dried to give a compound of formula (X).

A compound of formula (X) may also be prepared by reaction of a compoundof formula (XIA):

wherein T is a fluorine atom, with a suitable protecting agent, forexample a silylating agent such as N,O-bis(trimethylsilyl)acetamide,followed by reaction of the protected compound of formula (XIA) with acompound of formula (XVE):

P^(U)-E  (XVE)

wherein P^(U) is a suitable precursor to the protecting group P, forexample a 3,4-dihydro-2H-pyranyl group and E is an acyloxy group, forexample an acetate group.

For example, a suitable protecting agent, for exampleN,O-bis(trimethylsilyl)acetamide is added to a stirred suspension of acompound of formula (XIA), for example 2-fluoro-1H-purin-6-amine, in asuitable anhydrous solvent, for example anhydrous acetonitrile, and theresulting mixture heated to reflux for a suitable period of time, forexample 2-3 hours. The reaction mixture is then cooled to a suitabletemperature, for example 0-5° C. A solution of a compound of formula(XVE), for example tetrahydropyranyl acetate, in a suitable anhydroussolvent, for example anhydrous acetonitrile, is then added slowlyfollowed by the dropwise addition of a Lewis acid, for exampletrimethylsilyl trifluoromethanesulfonate. The reaction temperature isadjusted to a suitable temperature, for example 8-15° C., and stirringmaintained for a further period of time, for example 1-2 hours. Themixture is then quenched by addition of 1M sodium carbonate. The organiclayer is cooled to 0° C. with stirring. The precipitated solid is thencollected by, for example, filtration and dried.

A compound of formula (XI) may be prepared by reaction of a compound offormula (XII) with a compound of formula (XV).

For example, to a compound of formula (XII) is added a suitable organicsolvent, for example ethyl acetate, followed by p-toluenesulfonic acid.The mixture is heated to a suitable temperature, for example 50-60° C.,and then 3,4-dihydro-2H-pyran added. The reaction mixture is then heatedat a suitable temperature, for example 50-60° C. for a suitable periodof time, for example 4-5 hours. The solvent is then removed from thereaction mixture under reduced pressure to yield a compound of formula(XI).

Abbreviations

The following list provides definitions of certain abbreviations as usedherein. It will be appreciated that the list is not exhaustive, but themeaning of those abbreviations not herein below defined will be readilyapparent to those skilled in the art.

-   DCM Dichloromethane-   DMF N,N-Dimethylformamide-   DMSO Dimethylsulphoxide-   EtOAc Ethyl acetate-   Et₂O Diethyl ether-   HCl Hydrochloric acid-   HPLC High performance liquid chromatography-   ISCO Companion Automated flash chromatography equipment with    fraction analysis by UV absorption available from Presearch Limited,    Basingstoke, Hants., RG24 8PZ, UK-   MDAP HPLC Reverse phase HPLC on a C₁₈ column using a two-solvent    gradient and analysis of the fractions by electrospray mass    spectroscopy.-   SPE Solid phase extraction-   MeOH Methanol-   mins minutes-   Stripped Removal of solvent under reduced pressure-   TFA Trifluoroacetic acid-   iPr iso-Propyl-   t-Bu tert-Butyl-   Ms Mesyl-   Ac Acetyl-   n-Bu n-Butyl-   Ph Phenyl-   rt room temperature

The synthetic processes hereinbefore described are summarised in Scheme1.

Typical reaction conditions for each of the synthetic steps of Scheme 1are provided below:

-   A Dihydropyran/paratoluene sulphonic acid, e.g. 50° C. for 3-6    hours.-   A1 Dihydropyran/paratoluene sulphonic acid, e.g. 50° C. for 1 hour,    then ammonia/iPrOH, e.g. 60° C. for 4 hours, then add water and cool    to ambient temperature over 12-18 hours.-   A2 BSA in MeCN, reflux, cool to 0° C., then THP acetate in MeCN,    warm to 10° C., then NaHCO₃ (aq.)-   B Ammonia/iPrOH, e.g. 50° C. for 5 hours, then ambient temperature    for 12-18 hours, then 50° C. for 9 hours.-   C For X═NH, R^(A)═C₁₋₆alkyl: R^(A)NH₂/ethylene glycol e.g. 120° C.    for 12-18 hours.    -   For Z═O, R^(A)═C₁₋₆alkyl: R^(A)ONa/BuOH/dimethoxyethane e.g.        93-110° C. for 12-18 hours.-   C1 NBS in CHCl₃ e.g. 0-5° C. for 30 minutes then ambient temperature    for 0.5-1 hour, then e.g. NaOMe/methanol under N₂/60-70′C/12-18    hours, then TFA/MeOH e.g. ambient temperature for 18-65 hours.-   D NBS in CHCl₃ e.g. 0-5° C. for 30 minutes then ambient temperature    for 36-48 hours.-   E NaOMe/MeOH e.g. reflux 4-6 hours.-   F TFA/MeOH e.g. ambient temperature for 18-65 hours.-   G K₂CO₃/DMF then 50° C. for 1-1.5 hours, then add (VI), stir 40 min,    then add (IV)/Et₃N, then ambient temperature for 18 hours.-   G1 K₂CO₃/DMF, then 50° C. under N₂ for 30 minutes, then ambient    temperature, add (VI), stir for 20 hours.-   G2 Solution in DMF with N,N-diisopropylethylamine, then 50° C. for    48 hours, then more (IV) added then further 50° C. for 48 hours.-   H HCl/methanol, then ambient temperature for 18 hours.

Compounds of formulae (IV), (VI), (XIA), (XII), (XIII), (XIV), and (XV),are either known in the literature or are commercially available, forexample from Sigma-Aldrich, UK, or may be prepared by analogy with knownprocedures, for example those disclosed in standard reference texts ofsynthetic methodology such as J. March, Advanced Organic Chemistry, 6thEdition (2007), WileyBlackwell, or Comprehensive Organic Synthesis(Trost B. M. and Fleming I., (Eds.), Pergamon Press, 1991), eachincorporated herein by reference as it relates to such procedures.

Examples of other protecting groups that may be employed in thesynthetic routes described herein and the means for their removal can befound in T. W. Greene ‘Protective Groups in Organic Synthesis’, 4thEdition, J. Wiley and Sons, 2006, incorporated herein by reference as itrelates to such procedures.

For any of the hereinbefore described reactions or processes,conventional methods of heating and cooling may be employed, for exampletemperature-regulated oil-baths or temperature-regulated hot-blocks, andice/salt baths or dry ice/acetone baths respectively. Conventionalmethods of isolation, for example extraction from or into aqueous ornon-aqueous solvents may be used. Conventional methods of drying organicsolvents, solutions, or extracts, such as shaking with anhydrousmagnesium sulphate, or anhydrous sodium sulphate, or passing through ahydrophobic frit, may be employed. Conventional methods of purification,for example crystallisation and chromatography, for example silicachromatography or reverse-phase chromatography, may be used as required.Crystallisation may be performed using conventional solvents such asethyl acetate, methanol, ethanol, or butanol, or aqueous mixturesthereof. It will be appreciated that specific reaction timestemperatures may typically be determined by reaction-monitoringtechniques, for example thin-layer chromatography and LC-MS.

Where appropriate individual isomeric forms of the compounds of theinvention may be prepared as individual isomers using conventionalprocedures such as the fractional crystallisation of diastereoisomericderivatives or chiral high performance liquid chromatography (chiralHPLC).

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography.

Aspects of the invention are illustrated by reference to, but are in noway limited by, the following Examples.

General Experimental Details

Compounds were named using ACD/Name PRO 6.02 chemical naming softwarefrom Advanced Chemistry Developments Inc., Toronto, Ontario, M5H2L3,Canada.

Experimental details of LCMS systems A-D as referred to herein are asfollows:

System A

-   Column: 50 mm×2.1 mm ID, 1.7 m Acquity UPLC BEH C₁₈-   Flow Rate: 1 mL/min.-   Temp.: 40° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and negative mode electrospray ionisation.-   Solvents: A: 0.1% v/v formic acid in water    -   B: 0.1% v/v formic acid in acetonitrile

Gradient: Time (min.) A % B % 0 97 3 0.1 97 3 1.4 0 100 1.9 0 100 2.0 973

System B

-   Column: 30 mm×4.6 mm ID, 3.5 m Sunfire C₁₈ column-   Flow Rate: 3 mL/min.-   Temp: 30° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and negative mode electrospray ionisation-   Solvents: A: 0.1% v/v solution of formic acid in water    -   B: 0.1% v/v solution of formic acid in acetonitrile

Gradient: Time (min.) A % B % 0 97 3 0.1 97 3 4.2 0 100 4.8 0 100 4.9 973 5.0 97 3

System C

-   Column: 50 mm×2.1 mm ID, 1.7 m Acquity UPLC BEH C₁₈-   Flow Rate: 1 mL/min.-   Temp: 40° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and negative mode electrospray ionisation-   Solvents: A: 10 mM ammonium bicarbonate in water adjusted to pH10    with ammonia solution    -   B: acetonitrile

Gradient: Time (min.) A % B % 0 99 1 1.5 3 97 1.9 3 97 2.0 0 100

System D

-   Column: 50 mm×4.6 mm ID, 3.5 m XBridge C₁₈ column-   Flow Rate: 3 mL/min.-   Temp: 30° C.-   UV detection range: 210 to 350 nm-   Mass spectrum: Recorded on a mass spectrometer using    alternative-scan positive and negative mode electrospray ionisation-   Solvents: A: 10 mM ammonium bicarbonate in water adjusted to pH10    with ammonia solution    -   B: acetonitrile

Gradient: Time (min.) A % B % 0 99 1 0.1 99 1 4.0 3 97 5.0 3 97

Chromatographic purification was typically performed using pre-packedsilica gel cartridges. The Flashmaster II is an automated multi-userflash chromatography system, available from Argonaut Technologies Ltd,which utilises disposable, normal phase, Solid Phase Extraction (SPE)cartridges (2 g to 100 g). It provides quaternary on-line solvent mixingto enable gradient methods to be run. Samples are queued using themulti-functional open access software, which manages solvents,flow-rates, gradient profile and collection conditions. The system isequipped with a Knauer variable wavelength UV-detector and two GilsonFC204 fraction-collectors enabling automated peak cutting, collectionand tracking.

Solvent removal using a stream of nitrogen was performed at 30-40° C. ona GreenHouse Blowdown system available from Radleys DiscoveryTechnologies Saffron Walden, Essex, CB11 3AZ, UK

¹H NMR spectra were recorded in either CDCl₃ or DMSO-d₆ on either aBruker DPX 400 or Bruker Avance DRX or Varian Unity 400 spectrometer allworking at 400 MHz. The internal standard used was eithertetramethylsilane or the residual protonated solvent at 7.25 ppm forCDCl₃ or 2.50 ppm for DMSO-d₆.

Mass directed autopreparative HPLC was undertaken under the conditionsgiven below. The UV detection was an averaged signal from wavelength of210 nm to 350 nm and mass spectra were recorded on a mass spectrometerusing alternate-scan positive and negative mode electrospray ionization.

Method A

Method A was conducted on an XBridge C₁₈ column (typically 150 mm×19 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=10 mM aqueous ammonium bicarbonate adjusted to pH 10 with ammoniasolution.B=acetonitrile.

Method B

Method B was conducted on a Sunfire C₁₈ column (typically 150 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of formic acid in waterB=0.1% v/v solution of formic acid in acetonitrile.

Method C

Method C was conducted on a Sunfire C₁₈ column (typically 150 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of trifluoroacetic acid in waterB=0.1% v/v solution of trifluoroacetic acid in acetonitrile.

Method D

Method D was conducted on an Atlantis C₁₈ column (typically 100 mm×30 mmi.d. 5 μm packing diameter) at ambient temperature. The solventsemployed were:

A=0.1% v/v solution of formic acid in waterB=0.1% v/v solution of formic acid in acetonitrile.

Method E

Method E was conducted on a Supelcosil ABZ+Plus column (typically 100mm×21.2 mm i.d. 5 μm packing diameter) at ambient temperature. Thesolvents employed were:

A=0.1% v/v solution of formic acid in waterB=acetonitrile: water 95:5+0.05% formic acid

EXAMPLES Intermediate 1:2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine

To 2,6-dichloropurine (25.0 g) (available from, for example, Aldrich,UK) was added ethyl acetate (260 ml), followed by p-toluenesulfonic acid(0.253 g). The mixture was heated to 50° C. and then3,4-dihydro-2H-pyran (16.8 g) was added. The reaction mixture was thenheated at 50° C. for 4 hours. The reaction mixture was evaporated invacuo to give the title compound as a yellow solid (36.9 g).

1H NMR (CDCl₃): 8.35 (1H, s), 5.77 (1H, dd), 4.20 (1H, m), 3.79 (1H, m),2.20-1.65 (6H, m).

Intermediate 2: 2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2,6-Dichloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purine (36.9 g) was heatedwith 2M ammonia in isopropanol (250 ml) at 50° C. for 5 hours. Afterstanding at ambient temperature overnight, a further quantity of 2Mammonia in isopropanol (100 ml) was added to break up the resultant cakeand the reaction mixture was heated for a further 9 hours until thereaction was complete. To the reaction mixture was added water (70 ml)and the yellow solid filtered off. The solid was washed with isopropylalcohol:water (5:1 (v/v), 60 ml) and then air-dried under suction togive a first crop. The filtrate was re-filtered after standing overnightto isolate precipitate and both solids were dried in vacuo. The firstcrop was pure with the second crop material showing a very minorimpurity (isolated broad signal 3.5 ppm not seen in first crop) but wasotherwise identical. Solid first crop (28.4 g), solid second crop (3.42g).

1H NMR (CDCl₃): 8.01 (1H, s), 5.98 (2H, broad s), 5.70 (1H, dd), 4.16(1H, m), 3.78 (1H, m), 2.15-1.60 (6H, overlapping m).

Intermediate 2 (alternative method):2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To a solution of 2,6-dichloropurine (25 g) (available from, for example,Aldrich, UK) in dry ethyl acetate (200 ml) was added p-toluenesulfonicacid monohydrate (235 mg). The reaction was heated to 50° C. and3,4-dihydro-2H-pyran (18.1 ml) was added in one go. The reaction wasallowed to stir at 50′C for 1 hour and the solvent was removed underreduced pressure. This afforded a yellow solid. A suspension of thissolid (˜36 g) in 2.0M ammonia in isopropanol (460 ml) was heated undernitrogen at 60° C. for 4 hours with an attached condenser. The reactionwas poured into water (50 ml) and left to cool overnight. Theprecipitate was filtered and dried on a rotary evaporator (60° C.) for30 min. to afford the title compound as an off-white solid, 31 g (93%, 2steps).

MS calcd for (C₁₀H₁₂ClN₅O)⁺=254, 256

MS found (electrospray): (M)⁺=254, 256 (3:1)

¹H NMR ((CD₃)₂SO): δ 8.43 (1H, s), 7.82 (2H, s), 5.55 (1H, dd), 4.00(1H, m), 3.69 (1H, m), 2.21 (1H, m), 1.95 (2H, m), 1.74 (1H, m), 1.56(2H, m).

Intermediate 3:2-(Butyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To butan-1-ol (76 mL) was added portion wise sodium tert-butoxide (15.2g) (Note: reaction mixture gets warm). The above was stirred untilhomogeneous (ca.15 min) before2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (10.0 g) was thenadded to the resultant pale yellow solution. The reaction mixture wasthen heated to 100° C., overnight. The reaction mixture was stripped toremove as much butan-1-ol as possible before being partitioned betweendiethyl ether and water. The diethyl ether phase was separated and theaqueous re-extracted further with diethyl ether. Combined organic layersdried over magnesium sulphate (anhydrous). Magnesium sulphate wasfiltered off and filtrate stripped to give brown viscous oil which wasazeotroped with toluene (3 times) and placed under high vacuumovernight, transferred to new flask with dichloromethane and stripped,placed under high vacuum to give the title compound as a brown glass(9.45 g).

1H NMR (CDCl₃): 7.85 (1H, s), 5.92 (2H, broad s), 5.64 (1H, d), 4.32(2H, t), 4.14 (1H, m), 3.75 (1H, m), 2.10-1.95 (3H, overlapping m),1.81-1.58 (5H, overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 4:8-Bromo-2-(butyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

2-(Butyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (9.45 g) wasdissolved in chloroform (50 ml) and cooled to 0° C. (ice-bath). To thissolution was added portion wise N-bromosuccinimide (6.07 g) keeping thetemperature below 3° C. This gave a dark green solution, stirred at 2.5°C. for 30 mins. before allowing to warm to room temperature and thenstirring for 6 hours. The reaction mixture was then washed with water(100 ml, twice). Organic phase was dried/separated using a hydrophobicfrit and evaporated to give a dark brown gum which was purified bysilica chromatography (120 g) (ISCO) using a gradient elution of 0-50%ethyl acetate: cyclohexane to afford the title compound as a pale yellowsolid (8.37 g).

1H NMR (CDCl₃): 5.61 (1H, dd), 5.49 (2H, broad s), 4.32 (2H, m), 4.17(1H, m), 3.71 (1H, m), 3.04 (1H, m), 2.11 (1H, broad d), 1.89-1.45 (6H,overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 5:2-(Butyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-(butyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (8.37g) was heated to reflux with 25% sodium methoxide in methanol (14.4 ml)and methanol (65 ml) for 4.5 hours. The reaction mixture wasconcentrated under reduced pressure and partitioned between ethylacetate and saturated ammonium chloride solution. Separated organicphase and repeated extraction into ethyl acetate. Combined organicphases and washed with brine (twice). The organic phase was passedthrough a hydrophobic frit after separating aqueous and was evaporatedto give a light brown gum which was placed under high vacuum to give afoam (7.52 g) which collapsed to a gum (7.34 g) at ambient pressure andsolidified overnight to give the title compound as a yellow amorphoussolid.

MS calcd for (C₁₅H₂₃N₅O₃)⁺=321

MS found (electrospray): (M+H)⁺=322

1H NMR (CDCl₃): 5.50 (1H, dd), 5.17 (2H, broad s), 4.29 (2H, t), 4.12(3H, s and 1H, m), 3.70 (1H, m), 2.77 (1H, m), 2.05 (1H, m), 1.82-1.63(6H, overlapping m), 1.50 (2H, m), 0.97 (3H, t).

Intermediate 6: 2-(Butyloxy)-8-(methyloxy)-9H-purin-6-aminetrifluoroacetate salt

To a solution of2-(butyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(7.34 g) in methanol (100 ml) was added trifluoroacetic acid (10 ml).The mixture was stirred at ambient temperature over the weekend to givea suspension. The reaction mixture was concentrated to a small volume(thick slurry) before being diluted with ethyl acetate (50 ml). Theresultant slurry was filtered and washed with a small volume of ethylacetate until the filtrate was colourless. The solid remaining was driedby air and then in vacuo to give the title compound as a white solid(6.20 g). The filtrate obtained previously was concentrated to give aslurry which was diluted with a small volume of ethyl acetate (10 ml)and then filtered and dried as above. This second crop was isolated as awhite solid (0.276 g). Both crops were identical by NMR.

MS calcd for (C₁₀H₁₅N₅O₂)⁺=237

MS found (electrospray): (M+H)⁺=238

1H NMR (CD₃OD): 4.47 (2H, t), 4.15 (3H, s), 1.80 (2H, m), 1.50 (2H, m),0.99 (3H, t) (exchangeable NH₂, NH and COOH protons not observed).

Intermediate 7:N²—Butyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

To a solution of 2-chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(10 g) in dry ethylene glycol (50 ml) at room temperature and undernitrogen was added n-butylamine (16 ml) in one go. The reaction washeated at 120° C. overnight. The reaction was cooled to roomtemperature, diluted with ethyl acetate (150 ml) and washed with water(2×50 ml). The organic layer was dried over MgSO₄, filtered andconcentrated in vacuo. This afforded the title compound as a viscousgreen oil (10.2 g) that was used in the next step without furtherpurification.

MS calcd for (C₁₄H₂₂N₆O)⁺=290

MS found (electrospray): (M+H)⁺=291

¹H NMR ((CD₃)₂SO): δ 7.8 (1H, s), 6.6 (2H, s), 6.2 (1H, t), 5.4 (1H,dd), 4.0 (1H, m), 3.6 (1H, m), 3.2 (2H, m), 2.2 (1H, m), 1.9 (1H, m),1.8 (1H, m), 1.7 (1H, m), 1.5 (2H, m), 1.4 (2H, m), 1.3 (2H, m), 0.9(3H, t).

Intermediate 8: N²—Butyl-8-(methyloxy)-9H-purine-2,6-diamineTrifluoroacetic Acid Salt

To a solution of crudeN²-butyl-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine (ca.10.2 g)in dry chloroform (100 ml) at room temperature was addedN-bromosuccinimide (6.3 g) in portions over 5 mins. The dark solutionwas allowed to stir at room temperature for 30 mins. The reactionmixture was washed with water (20 ml). The organic phase was passedthrough a hydrophobic frit and concentrated in vacuo. This afforded abeige solid which was dissolved in dry methanol (100 ml) and at roomtemperature under nitrogen was added sodium methoxide solution (25 wt. %in methanol, 24 ml) in one go. The reaction was heated at 65° C., with acondenser attached, overnight. The reaction was cooled and concentratedin vacuo. The resultant orange residue was taken up in ethyl acetate(150 ml) and poured into saturated aqueous ammonium chloride (50 ml).The organic layer was separated and washed further with water (50 ml).The organic layer was dried over MgSO₄, filtered and concentrated invacuo. To this material in dry methanol (70 ml) at room temperature wasadded trifluoroacetic acid (7 ml) in one go. The reaction was stirredfor 30 hours and concentrated in vacuo to yield a dark brown solid. Thiswas taken up in diethyl ether (20 ml) and triturated. The solid wasfiltered to afford the title compound as a beige solid (3.3 g, 35%, 4steps).

MS calcd for (C₁₀H₁₆N₆O)⁺=236

MS found (electrospray): (M+H)⁺=237

¹H NMR ((CD₃)₂SO): δ 13.3-12.3 (1H, br.m), 8.6-7.3 (2H, m), 4.05 (3H,s), 3.28 (2H, m), 1.52 (2H, m), 1.33 (2H, m), 0.89 (3H, t) (remainingexchangeable protons not clear).

Intermediate 9:2-{[(1S)-1-Methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Method A

Sodium t-butoxide (48.5 g, 505 mmol) was added portionwise to(S)-2-pentanol (185 ml) (available from, for example, Julich ChiralSolutions, Germany) at room temperature stirred until homogeneous (Note:reaction is exothermic).2-Chloro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (32 g, 126 mmol)was added and the reaction mixture heated at 70° C. for 72 hours. Thereaction was cooled to room temperature and partitioned between ethylacetate (500 ml) and water (500 ml). The organic phase was washed withsaturated sodium chloride solution (100 ml), dried (MgSO₄), filtered andevaporated. The residue was triturated with ether and the solid materialfiltered. The precipitate was re-washed with ether and the filtratescombined and evaporated. The crude material (ca. 30 g) was dissolved inDMSO:methanol (1:1) and purified by chromatography on a reverse phase(C₁₈) column (330 g) using a gradient of 25-65% acetonitrile (+0.1%TFA)-water(+0.1% TFA) over 8 column volumes, the fractions wereimmediately neutralised with saturated aqueous sodium carbonatesolution. Appropriate fractions were combined and partitioned betweendichloromethane and saturated aqueous sodium hydrogen carbonate. Theorganic phase was dried by passage through a hydrophobic frit, filteredand evaporated to give the title compound as a pale cream foam (14.97g).

LCMS (System B): t_(RET)=2.21 min; MH⁺306

Method B

Sodium t-butoxide (206 g, 2.144 mol) was added to (S)-2-pentanol (720ml, 6.58 mol) (available from, for example, Julich Chiral Solutions,Germany) in a 2 L round bottomed flask. The mixture was stirred at 50°C. until all the sodium t-butoxide had dissolved.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (130 g, 548 mmol)was then added in portions over 5 mins. After 3 hours LCMS analysisindicated complete consumption of the starting material and the mixturewas poured into ice/water (3 L) and then extracted with methyl t-butylether. This resulted in emulsion formation and the mixture was filteredthrough Celite and the organic phase was separated. The aqueous layerwas then treated with solid NaCl and then re-extracted with methylt-butyl ether. The organic extracts were combined and washed with brine,dried over magnesium sulfate, filtered and then evaporated to yield thetitle compound as a pale brown gum (158.59 g).

LCMS (System D): t_(RET)=2.65 min; MH⁺306

Intermediate 10:8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (12.16 g, 68.3 mmol) was added portionwise over 5mins. to a stirred solution of 2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(14.9 g, 48.8 mmol) in chloroform (80 ml) at <5° C. under an atmosphereof nitrogen. The reaction mixture was stirred at <5° C. for 5 hours thenwashed with saturated sodium hydrogen carbonate solution (80 ml) thenwater (80 ml). The foam was dissolved in DCM (50 ml) and washed withwater (50 ml) then brine (50 ml). The combined aqueous phases werewashed with DCM (50 ml). The combined organic layers were dried througha hydrophobic frit, and the solvent removed in vacuo to yield the titlecompound as an orange foam (18.5 g).

LCMS (System D): t_(RET)=3.06 min; MH⁺384/386

Intermediate 11: 2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-{[(1S)-1-methylbutyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (7.1g, 18.48 mmol) was dissolved in anhydrous methanol (70 ml) and asolution of sodium methoxide (25%) in methanol (8 ml) was added dropwiseunder an atmosphere of nitrogen. The solution was heated to reflux at90° C. for 4 hours under an atmosphere of nitrogen. Additional sodiummethoxide in methanol (25% solution, 3 ml) was added and the reactionwas stirred at 60° C. for a further 16 hours. An additional portion ofsodium methoxide in methanol (25% solution, 5 ml) was added and thereaction was stirred at 90° C. for a further 7 hours. The solvent wasremoved on the rotary evaporator and the crude product was partitionedbetween EtOAc (75 ml) and saturated ammonium chloride solution (75 ml).The organic layer was washed with brine (75 ml). The solvent was removedon the rotary evaporator to yield the title compound as a pale orangefoam (6 g).

LCMS (System C): t_(RET)=1.14 min; MH⁺336, 337

Intermediate 12: 2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine TrifluoroacetateSalt

2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(6 g, 17.89 mmol) was dissolved in methanol (50 ml). Trifluoroaceticacid (20.67 ml, 268 mmol) was added dropwise, and the mixture stirred at20° C. for 72 hours under an atmosphere of nitrogen. The solvent wasremoved in vacuo, and the resulting solid was washed with ethyl acetateand filtered. The filtrate was stripped and the residue washed withethyl acetate. The combined solid residues were dried in the vacuum ovenfor 2 hours to give the title compound as an off white solid (5.3 g).

LCMS (System C): t_(RET)=0.76 min; MH⁺252, 253

Intermediate 13:2-(Butyloxy)-9-(3-chloropropyl)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-9H-purin-6-amine trifluoroacetate (4.7 g,13.38 mmol) and potassium carbonate (4.62 g, 33.4 mmol) in dry DMF (50ml) were stirred and heated at 50° C., under nitrogen, for 75 mins. Themixture was allowed to cool to room temperature and then cooled to 0° C.and 1-bromo-3-chloropropane (2.106 g, 13.38 mmol) was added. The mixturewas stirred at 0 to 10° C. for approximately 5 hours then allowed towarm to room temperature and stirred for approximately a further 40hours when LCMS indicated approximately 70% of the desired product. Themixture was allowed to settle and the supernatant was pipetted off andthe solvent evaporated on a rotary evaporator using a high vacuum pumpat about 23° C. Chloroform and water was added to the combined residueswhich were stirred and the phases separated using a hydrophobic frit.The aqueous layer was re-extracted with further portions of chloroformand the combined chloroform extracts were evaporated under high vacuumat 23° C. to give a yellow solid (2.798 g). This crude material wascombined with similar material obtained from two similar preparations(0.56 g and 0.995 g) and purified by flash column chromatography onsilica using 2:1 ethyl acetate/chloroform as eluant to give the titlecompound as an off-white solid. (3.011 g).

LCMS (System D): t_(RET)=2.79 min; MH⁺314, 316

Intermediate 14:2-(Butyloxy)-9-(4-chlorobutyl)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-9H-purin-6-amine trifluoroacetate (2 g, 5.69mmol) and potassium carbonate (1.967 g, 14.23 mmol) were suspended inDMF (20 ml) and heated to 50° C., under nitrogen for 30 mins. Themixture was cooled to room temperature, 1-bromo-4-chlorobutane (0.656ml, 5.69 mmol) was added and stirring continued at room temperature for20 hours. The solvent was evaporated under reduced pressure and theresidue was partitioned between DCM (40 ml) and water (40 ml). Thelayers were separated using a hydrophobic frit and the aqueous layerwashed with DCM (10 ml). The combined organic extracts were concentratedin vacuo to give crude material that was purified by silicachromatography using the FlashMaster (70 g cartridge) eluting with acyclohexane:ethyl acetate 0-100% gradient over 30 mins. Theproduct-containing fractions were combined and evaporated to give thetitle compound as a white solid (1.4 g).

LCMS (System D): t_(RET)=2.92 min; MH⁺=328, 330

Intermediate 15:2-(Butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-9H-purin-6-amine trifluoroacetate (2 g, 5.69mmol) and potassium carbonate (1.967 g, 14.23 mmol) were suspended inDMF (20 ml) and heated to 50° C., under nitrogen for 1 hour. The mixturewas cooled to room temperature, 1-bromo-5-chloropentane (0.75 ml, 5.69mmol) was added and stirring was continued at room temperature for 18hours. The reaction mixture was partitioned between DCM (40 ml) andwater (40 ml) and the layers were separated using a hydrophobic frit.The aqueous layer was extracted again with DCM (10 ml) and the combinedorganics were washed with saturated lithium chloride solution, separated(hydrophobic frit) and concentrated in vacuo to give the title compoundas a yellow oil (1.946 g).

LCMS (System B): t_(RET)=2.58 min; MH⁺=342, 344

Intermediate 16:2-(Butyloxy)-9-(5-chlorohexyl)-8-(methyloxy)-9H-purin-6-amine

To a solution of 2-(butyloxy)-8-(methyloxy)-9H-purin-6-aminetrifuoroacetate salt (3 g, 8.54 mmol) in DMF (30 ml) was added potassiumcarbonate (2.95 g, 21.35 mmol) and the mixture stirred at 60° C. for 1hour under an atmosphere of nitrogen. The mixture was then cooled toroom temperature and 1-bromo-6-chlorohexane (1.27 ml, 8.54 mmol) wasadded and the reaction heated to 50° C. and stirred overnight under anatmosphere of nitrogen. The reaction mixture was diluted with water (ca.50 ml) and extracted with ethyl acetate (2×70 ml). The combined organicextracts were dried (MgSO₄), filtered and the filtrate concentrated togive an orange oil (ca.3.5 g). This material was dissolved indichloromethane and purified on a Flashmaster II (70 g aminopropylcartridge) using a 0-100% ethyl acetate in cyclohexane gradient over 60mins. The appropriate fractions were combined and evaporated in vacuo togive the title compound as a yellow oil which solidified to a paleyellow solid (1.2 g).

LCMS (System D): t_(RET)=3.59 min; MH⁺=356, 358

Intermediate 17:N²-Butyl-9-(3-chloropropyl)-8-(methyloxy)-9H-purine-2,6-diamine

N²-Butyl-8-(methyloxy)-9H-purine-2,6-diamine trifluoroacetate (701 mg,2.001 mmol) and potassium carbonate (690 mg, 4.99 mmol) were suspendedin DMF (10 ml) and the mixture heated at 50° C. under nitrogen for 2hours. The mixture was allowed to cool and then 1-bromo-3-chloropropane(198 μl, 2.002 mmol) was added and the reaction mixture stirred atambient temperature overnight. After 16 hours the reaction mixture waspartitioned between water and DCM (25 ml of each). The aqueous phase wasextracted with further DCM (2×20 ml). The combined DCM extracts weredried over magnesium sulphate and concentrated in vacuo to give theimpure title compound as a pale yellow oil with some solid present (0.76g) which was used without further purification.

LCMS (System D): t_(RET)=2.75 min; MH⁺=313, 315

Intermediate 18:N²-Butyl-9-(4-chlorobutyl)-8-(methyloxy)-9H-purine-2,6-diamine

N²-Butyl-8-(methyloxy)-9H-purine-2,6-diamine trifluoroacetate (5 g,14.27 mmol) and potassium carbonate (4.93 g, 35.7 mmol) were suspendedin DMF (40 ml) and heated to 50° C. under nitrogen for 30 mins. Themixture was cooled to room temperature, 1-bromo-4-chlorobutane (1.645ml, 14.27 mmol) was added and stirring was continued at room temperaturefor 20 hours. The solvent was concentrated under vacuum and the residuewas partitioned between DCM (100 ml) and water (100 ml). The layers wereseparated using a hydrophobic frit and the aqueous phase wasre-extracted with DCM (100 ml). The combined organics extracts wereconcentrated in vacuo and the residue purified by chromatography using aFlashMaster apparatus (100 g silica cartridge) and using a DCM:methanol0-25% gradient over 40 mins. The desired fractions were combined andconcentrated under vacuum to give the impure title compound as a yellowoil (5.1 g).

LCMS (System D): t_(RET)=2.88 min; MH⁺=327, 329

Intermediate 19: 9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

2-{[(1 S)-1-Methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-aminetrifluoroacetate (600 mg, 1.642 mmol) and potassium carbonate (567 mg,4.11 mmol) were stirred at 60° C. in DMF (10 ml) for 1 hour undernitrogen. The reaction was cooled to room temperature when1-bromo-5-chloropentane (0.216 ml, 1.642 mmol) and triethylamine (0.343ml, 2.464 mmol) were added and the mixture stirred at 20° C. undernitrogen for 16 hours. The mixture was then diluted with water (10 ml)and brine (10 ml) and extracted with DCM (2×10 ml). The combined organicextracts were evaporated and the residue dissolved in DCM and purifiedby column chromatography using the Flashmaster II (70 g aminopropylcartridge) with a 0-100% ethyl acetate in cyclohexane gradient over 40mins. The appropriate fractions were combined and evaporated in vacuo togive the title compound as a yellow gum (430 mg).

LCMS (System D): t_(RET)=4.15 min; MH⁺=356, 358

Intermediate 20:9-[3-(1-Azetidinyl)propyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate (100 mg,0.285 mmol) was dissolved in DMF (1 ml) and potassium carbonate (98 mg,0.712 mmol) was added. The reaction mixture was stirred at 50° C. undernitrogen for 1 hour and then cooled to room temperature.1,3-Dibromopropane (0.029 ml, 0.285 mmol) was added and after stirringfor a further 40 mins. azetidine (0.038 ml, 0.569 mmol) andtriethylamine (0.079 ml, 0.569 mmol) in DMF (1 ml) were added. Thereaction mixture was then stirred for a further 18 hours. The solventwas removed and the residue was partitioned between dichloromethane (2ml) and water (2 ml). The layers were separated using a hydrophobic fritand the aqueous phase was re-extracted with DCM (2 ml). The combinedorganic extracts were concentrated and the residue was dissolved in 1:1MeOH:DMSO (1 ml) and purified by MDAP (Method A). The product containingfractions were evaporated under a stream of nitrogen to give the titlecompound as a white solid (13 mg).

LCMS (System B): t_(RET)=1.07 min; MH⁺=335

Intermediate 21:2-(Butyloxy)-8-(methyloxy)-9-[3-(1-pyrrolidinyl)propyl]-9H-purin-6-amine

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,3-dibromopropane and pyrrolidine.

LCMS (System C): t_(RET)=0.60 min; MH⁺=349

Intermediate 22:2-(Butyloxy)-9-[3-(hexahydro-1H-azepin-1-yl)propyl]-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,3-dibromopropane and hexahydro-1H-azepine.

LCMS (System B): t_(RET)=1.24 min; MH⁺=377

Intermediate 23:9-[4-(1-Azetidinyl)butyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-9-(4-chlorobutyl)-8-(methyloxy)-9H-purin-6-amine (100 mg,0.305 mmol), azetidine (0.021 ml, 0.305 mmol) andN,N-diisopropylethylamine (0.107 ml, 0.610 mmol) were dissolved in DMF(2 ml) and heated at 50° C. for 48 hours. LCMS indicated the reaction tobe incomplete and additional azetidine (0.021 ml, 0.305 mmol) andN,N-diisopropylethylamine (0.107 ml, 0.610 mmol) were added and thereaction mixture heated at 50° C. for a further 48 hours. The mixturewas then partitioned between DCM (4 ml) and water (4 ml) and the layersseparated using a hydrophobic frit. The aqueous phase was re-extractedwith DCM (4 ml) and the combined organic extracts were concentrated andthe residue purified by MDAP (Method A). The product-containingfractions were evaporated under a stream of nitrogen to give the titlecompound as a clear gum (7.6 mg).

LCMS (System B): t_(RET)=1.15 min; MH⁺=349

Intermediate 24:2-(Butyloxy)-8-(methyloxy)-9-[4-(1-pyrrolidinyl)butyl]-9H-purin-6-amineFormic Acid Salt

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,4-dibromobutane and pyrrolidine but with mass directed autopreparationusing Method D.

LCMS (System B): t_(RET)=1.19 min; MH⁺=363

Intermediate 25:2-(Butyloxy)-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purin-6-amineFormic Acid Salt

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,4-dibromobutane and piperidine but with sequential mass directedautopreparations using Method A followed by Method D.

LCMS (System B): t_(RET)=1.22 min; MH⁺=377

Intermediate 26:2-(Butyloxy)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,4-dibromobutane and hexahydro-1H-azepine.

LCMS (System B): t_(RET)=1.30 min; MH⁺=391

Intermediate 27:9-[5-(1-Azetidinyl)pentyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine

2-(Butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine (100 mg,0.293 mmol), azetidine (0.020 ml, 0.293 mmol) andN,N-diisopropylethylamine (0.102 ml, 0.585 mmol) were dissolved in DMF(2 ml) and heated at 50° C. for 72 hours. The solvent was removed invacuo and the residue partitioned between DCM (5 ml) and water (5 ml)and the layers separated using a hydrophobic frit. The aqueous phase wasre-extracted with DCM (5 ml) and the combined organic extractsconcentrated and the residue dissolved in 1:1 MeOH:DMSO (1 ml) andpurified by MDAP (Method A). The product-containing fractions wereevaporated under a stream of nitrogen to give the title compound as aclear gum (6.8 mg).

LCMS (System B): t_(RET)=1.26 min; MH⁺=363

Intermediate 28:2-(Butyloxy)-8-(methyloxy)-9-[5-(1-pyrrolidinyl)pentyl]-9H-purin-6-amine

Prepared similarly to Intermediate 27 from2-(butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine andpyrrolidine.

LCMS (System B): t_(RET)=1.27 min; MH⁺=377

Intermediate 29:2-(Butyloxy)-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine

Prepared similarly to Intermediate 27 from2-(butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine andpiperidine.

LCMS (System B): t_(RET)=1.33 min; MH⁺=391

Intermediate 30:2-(Butyloxy)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 27 from2-(butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine andhexahydro-1H-azepine but with sequential purifications by MDAPs usingMethod A followed by Method E.

LCMS (System B): t_(RET)=1.38 min; MH⁺=405

Intermediate 31:2-(Butyloxy)-9-[5-(hexahydro-1(2H)-azocinyl)pentyl]-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 38 from2-(butyloxy)-9-(5-chloropentyl)-8-(methyloxy)-9H-purin-6-amine andoctahydroazocine.

LCMS (System B): t_(RET)=1.45 min; MH⁺=419

Intermediate 32:2-(Butyloxy)-8-(methyloxy)-9-[6-(1-pyrrolidinyl)hexyl]-9H-purin-6-amine

Prepared similarly to Intermediate 38 from2-(butyloxy)-9-(6-chlorohexyl)-8-(methyloxy)-9H-purin-6-amine andpyrrolidine.

LCMS (System D): t_(RET)=2.97 min; MH⁺=391

Intermediate 33:2-(Butyloxy)-8-(methyloxy)-9-[6-(1-piperidinyl)hexyl]-9H-purin-6-amine

Prepared similarly to Intermediate 38 from2-(butyloxy)-9-(6-chlorohexyl)-8-(methyloxy)-9H-purin-6-amine andpiperidine.

LCMS (System D): t_(RET)=3.12 min; MH⁺=405

Intermediate 34:2-(Butyloxy)-9-[6-(hexahydro-1H-azepin-1-yl)hexyl]-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 38 from2-(butyloxy)-9-(6-chlorohexyl)-8-(methyloxy)-9H-purin-6-amine andhexahydro-1H-azepine.

LCMS (System D): t_(RET)=3.20 min; MH⁺=419

Intermediate 35:N²-Butyl-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purine-2,6-diamine

N²-Butyl-8-(methyloxy)-3H-purine-2,6-diamine trifluoroacetate (192 mg,0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspendedin DMF (3 ml) and heated to 60° C. for 1 hour. The reaction mixture wascooled to room temperature, 1-bromo-4-chlorobutane (0.063 ml, 0.547mmol) added and the reaction stirred for a further 18 hours. Piperidine(0.054 ml, 0.547 mmol) and triethylamine (0.076 ml, 0.547 mmol) wereadded and the reaction mixture heated to 60° C. for 72 hours. Thesolvent was removed in vacuo and the residue partitioned between DCM (2ml) and water (2 ml). The aqueous phase was re-extracted with DCM (2 ml)and the combined organic extracts were concentrated. The residue (ca.200mg) was dissolved in 1:1 MeOH:DMSO (1 ml) and purified by MDAP (MethodA). The product containing fractions were evaporated in vacuo to givethe impure title compound as a yellow gum (106 mg) which was usedwithout further purification.

LCMS (System B): t_(RET)=1.11 min; MH⁺=376

Intermediate 36:N²—Butyl-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-8-(methyloxy)-9H-purine-2,6-diamine

N²-Butyl-8-(methyloxy)-3H-purine-2,6-diamine trifluoroacetate (192 mg,0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspendedin DMF (3 ml) and heated to 60° C. for 1 hour. The reaction mixture wascooled to room temperature, 1-bromo-4-chlorobutane (0.063 ml, 0.547mmol) was added and the reaction stirred for a further 18 hours.Hexahydro-1H-azepine (54.2 mg, 0.547 mmol) and triethylamine (0.076 ml,0.547 mmol) were added and the reaction mixture heated to 60° C. for 18hours. The solvent was removed in vacuo and the residue was partitionedbetween DCM (5 ml) and water (5 ml). The aqueous phase was re-extractedwith DCM (5 ml) and the combined organic extracts were concentrated invacuo. The residue was dissolved in 1:1 MeOH:DMSO (2 ml) and purified in2 injections by MDAP (Method B). This provided material (74 mg) that wasstill impure and which was repurified by MDAP (Method A). The productcontaining fractions were evaporated under a stream of nitrogen to givethe title compound as a clear gum (13 mg).

LCMS (System B): t_(RET)=1.12 min; MH⁺=390

Intermediate 37: 9-[4-(Hexahydro-1H-azepin-1-yl)butyl]-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 36 from 2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1-bromo-4-chlorobutane, and hexahydro-1H-azepine but with threesequential MDAPs using Method B followed by Method A (×2).

LCMS (System B): t_(RET)=1.41 min; MH⁺=405

Intermediate 38: 2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine

9-(5-Chloropentyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine (80 mg, 0.225mmol), triethylamine (0.031 ml, 0.225 mmol) and piperidine (0.045 ml,0.45 mmol) were suspended in DMF (3 ml) and the mixture heated to 70° C.for 18 hours. The solvent was removed and the residue partitionedbetween DCM (4 ml) and saturated sodium bicarbonate (4 ml). The aqueousphase was re-extracted with further DCM and the combined organicextracts were concentrated and the residue dissolved in 1:1 MeOH:DMSO (1ml) and purified by MDAP (Method A). The product-containing fractionswere combined and evaporated under a stream of nitrogen to give thetitle compound (47.2 mg).

LCMS (System D): t_(RET)=3.11 min; MH⁺=405

Intermediate 39: 2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N,O-bis(trimethylsilyl)acetamide (975 mL, 3.988 mol) was added to astirred suspension of 2-fluoro-1H-purin-6-amine (200 g, 1.306 mmol)(available from, for example, AlliedSignal, US) in anhydrousacetonitrile (4 L) in a 10 L controlled lab reactor and the resultingmixture heated to reflux and maintained at that temperature for 2 hours.The circulator was then re-programmed and the reaction mixture cooled to0° C. A solution of tetrahydropyranyl acetate (preparation described inTetrahedron Letters 2006, 47(27), 4741) (282 g, 1.959 mol) in anhydrousacetonitrile (500 ml) was then added slowly via a dropping funnelfollowed by trimethylsilyl trifluoromethanesulfonate (283 mL, 1.567 mol)dropwise via a dropping funnel. No significant exotherm was observed.The circulator temperature was re-adjusted to 10° C. and stirringmaintained for a further 1 hour. The mixture was then quenched byaddition of 1M sodium carbonate (4 L). A solid precipitate was observedand the pH checked to be basic. Additional water was added to thesuspension (1 L) and on standing the layers separated with the aqueouslayer containing significant solid inorganics. The majority of theaqueous and inorganic solid was separated. The organic layer stillcontained significant solid and was cooled to 0° C. with stirring toencourage further precipitation. The solid was the collected byfiltration and the pad was washed very well with water then dried invacuo at 40° C. overnight to give the title compound as a cream colouredsolid (152.8 g).

LCMS (System D): t_(RET)=1.71 min; MH⁺=238

Intermediate 40:2-{[(1S)-1-Methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Sodium tert-butoxide (3.24 g, 33.7 mmol) was added portionwise withstirring to (2S)-2-butanol (10 g, 135 mmol).2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (2 g, 8.43 mmol)was added to the resulting suspension and the mixture heated to 50° C.for 6 hours when LCMS showed complete reaction. After cooling themixture was diluted with ethyl acetate (100 ml), and washed with water(50 ml) and the aqueous layer extracted again with ethyl acetate (50ml). The combined organic extracts were washed with brine, dried using ahydrophobic frit and evaporated in vacuo (at 62° C. to remove the excessalcohol). The residue (2.52 g) was dissolved in dichloromethane andpurified on an aminopropyl cartridge (110 g) using a Flashmaster IIapparatus and eluting with a 0-100% ethyl acetate in cyclohexanegradient over 60 mins. The appropriate fractions were combined andevaporated in vacuo to give the title compound as a white solid (1.935g).

LCMS (System D): t_(RET)=2.41 min; MH⁺=292

Intermediate 41:8-Bromo-2-{[(1S)-1-methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (1.182 g, 6.64 mmol) was added portionwise to asolution of 2-{[(1S)-1-methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.935 g, 6.64 mmol) in chloroform (50 ml) at 0-5° C. The resultinggreen solution was stirred at 0-5° C. for 1 hour during which time itturned red and the mixture was then allowed to warm to room temperatureand stirred overnight. The resulting green solution was washed withwater (2×20 ml), separated using a hydrophobic frit and concentrated.The residue was dissolved in dichloromethane and purified by silica gelchromatography (100 g cartridge) using a Flashmaster II apparatus and a0-100% ethyl acetate-cyclohexane gradient over 60 mins. The appropriatefractions were combined and evaporated in vacuo to give the titlecompound as a yellow foam (1.79 g).

LCMS (System B): t_(RET)=2.58 min; MH⁺=370/372

Intermediate 42: 8-(Methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-{[(1S)-1-methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.79 g, 4.83 mmol) was dissolved in methanol (15 ml) and 25% sodiummethoxide in methanol (3.2 ml, 4.83 mmol) was added and the mixtureheated to reflux for 2.5 hours. The reaction mixture was left standingat room temperature overnight and then concentrated in vacuo and theresidue partitioned between dichloromethane (40 ml) and saturatedammonium chloride solution (40 ml). The layers were separated using ahydrophobic frit and the aqueous phase was re-extracted withdichloromethane (40 ml). The combined organic extracts were concentratedin vacuo to give the title compound as a yellow foam (1.65 g).

LCMS (System B): t_(RET) '² 2.11 min; MH⁺=322

Intermediate 43: 8-(Methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-1H-purin-6-amine trifluoroacetate

Prepared similarly to Intermediate 12 from 8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.19 min; MH⁺=238

Intermediate 44: 9-(4-Chlorobutyl)-8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9H-purin-6-amine

Prepared similarly to Intermediate 18 from 8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-1H-purin-6-amine trifluoroacetate and1-bromo-4-chlorobutane with purification on an aminopropyl (NH₂)cartridge using a 0-100% ethyl acetate-cyclohexane gradient.

LCMS (System D): t_(RET)=2.83 min; MH⁺=328/330

Intermediate 45:2-{[(1S)-1-Methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Sodium t-butoxide (4.86 g, 50.6 mmol) was added portionwise to a stirredmixture of (S)-2-hexanol (12 g, 117 mmol) and 1,2-dimethoxyethane (12ml). The resultant mixture was heated to 50° C. under an atmosphere ofnitrogen and then 2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(3 g, 12.65 mmol) was added. The resultant mixture was maintained at 50°C. for 20 hours when LCMS indicated complete reaction. The mixture wascooled to room temperature and partitioned between ethyl acetate (100ml) and water (100 ml). The organic phase was washed with water (100 ml)then saturated brine (50 ml), dried over anhydrous magnesium sulphate,filtered and evaporated. The residue was dissolved in dichloromethaneand purified on an aminopropyl (NH₂) cartridge (100 g) eluting with a0-100% ethyl acetate in cyclohexane gradient over 40 mins. Theappropriate fractions were combined and evaporated in vacuo to give thetitle compound as a white foam (1.665 g).

LCMS (System D): t_(RET)=2.88 min; MH⁺=320

Intermediate 46:8-Bromo-2-{[(1S)-1-methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (1.504 g, 8.45 mmol) was added portionwise to astirred solution of 2-{[(1S)-1-methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(2.453 g, 7.68 mmol) in chloroform (40 ml) under at atmosphere ofnitrogen cooled in an ice-bath. After 3 hours LCMS indicated thereaction to be 80% complete and more N-bromosuccinimide (0.68 g) wasadded and stirring continued for a further 2 hours. Water (40 ml) wasadded and the phases separated using a hydrophobic frit. The organicphase was evaporated and the residue dissolved in dichloromethane andpurified on an aminopropyl (NH₂) cartridge (100 g) using a 0-100% ethylacetate in cyclohexane gradient followed by a 0-20% methanol (+1%triethylamine) gradient over 60 mins. The appropriate fractions werecombined and evaporated in vacuo to the title compound as a white foam(2.38 g).

LCMS (System D): t_(RET)=3.24 min; MH⁺=398/400

Intermediate 47: 8-(Methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

A solution of sodium methoxide in methanol (0.5M, 20 ml, 10 mmol) wasadded to a solution of 8-bromo-2-{[(1S)-1-methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(2.368 g, 5.95 mmol) in methanol (10 ml) and the mixture heated underreflux for 5 hours. More sodium methoxide in methanol (4 ml, 2 mmol) wasadded and the mixture refluxed for a further 2 hours and then cooled andevaporated. The residue was partitioned between ethyl acetate (100 ml)and water (100 ml). The organic phase was separated, washed withsaturated brine, dried over anhydrous magnesium sulphate, filtered andevaporated. The residue was dissolved in dichloromethane and purified onan aminopropyl (NH₂) cartridge (100 g) using a 0-100% ethyl acetate incyclohexane gradient over 40 mins. The appropriate fractions werecombined and evaporated in vacuo to give the title compound as a whitefoam (1.725 g).

LCMS (System D): t_(RET)=3.06 min; MH⁺=350

Intermediate 48: 8-(Methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-1H-purin-6-amine trifluoroacetate

Trifluoroacetic acid (2.3 ml, 3.40 g, 29.9 mmol) was added to a stirredsolution of 8-(methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.479 g, 4.23 mmol) in methanol (25 ml). The resultant mixture wasstirred for 66 hours under an atmosphere of nitrogen and then evaporatedand dried in vacuo to give the title compound as a white solid (1.65 g).

LCMS (System D): t_(RET)=2.14 min; MH⁺=266

Intermediate 49: 9-(4-Chlorobutyl)-8-(methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-9H-purin-6-amine

Prepared similarly to Intermediate 44 from 8-(methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-1H-purin-6-amine trifluoroacetate and1-bromo-4-chlorobutane.

LCMS (System D): t_(RET)=3.22 min; MH⁺=356/358

Intermediate 50:2-[(1-Methylethyl)oxy]-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Sodium t-butoxide (1.30 g, 13.53 mmol) was added to 2-propanol (16.95ml, 220 mmol) portionwise with stirring over 5 mins.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (2 g, 8.43 mmol)was added and the reaction mixture heated and stirred at 50° C. for 4hours and then allowed to cool to room temperature. The reaction mixturewas then diluted with ethyl acetate (75 ml), washed with water (3×25 ml)and the combined aqueous layers extracted again with ethyl acetate (2×25ml). The combined organic layers were dried by passage through ahydrophobic frit, filtered and evaporated to give an off-white solid(2.30 g). This material was dissolved in dichloromethane and purifiedusing an aminopropyl SPE cartridge (70 g) eluted with a 0-100% ethylacetate in cyclohexane gradient. The appropriate fractions were combinedand evaporated to give a white solid (1.6 g) which was further purifiedby column chromatography using a reverse phase (C₁₈) Flashmaster IIsystem loading in 1:1 MeOH/DMSO and eluting with 0-50% acetonitrile(+0.1% TFA) in water (+0.1% TFA) gradient over 40 mins. collectingfractions in vials containing ca. 2 mL of saturated aqueous sodiumbicarbonate solution. The appropriate fractions were combined, andextracted with dichloromethane (3×100 mL). The combined organic extractswere dried by passage through a hydrophobic frit and evaporated to givethe title compound as a white solid (888 mg).

LCMS (System B): t_(RET)=1.76 min; MH⁺=278

Intermediate 51:8-Bromo-2-[(1-methylethyl)oxy]-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (604 mg, 3.39 mmol) was added to a solution of2-[(1-methylethyl)oxy]-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(888 mg, 3.20 mmol) in chloroform (30 ml) at 0-5° C. under nitrogen. Themixture was stirred at 0-5° C. for 1 hour during which time it becamereddish brown in colour and it was then warmed to room temperature andstirred for a further 4 hours. LCMS indicated the reaction to beincomplete and more N-bromosuccinimide (114 mg, 0.641 mmol) was addedand the reaction mixture stirred at room temperature overnight. Thereaction mixture was then diluted with chloroform (30 ml), washed withwater (2×20 ml) and the layers were separated using a hydrophobic fritand the organic layer was evaporated to give a red solid (1.16 g). Thismaterial was dissolved in dichloromethane and purified by silica gelchromatography on an SPE cartridge (50 g) using a 0-100% ethyl acetatein cyclohexane gradient as eluent. The appropriate fractions werecombined and evaporated to give the title compound as a pale yellowsolid 712 mg.

LCMS (System B): t_(RET)=2.36 min; MH⁺=356/358

Intermediate 52:2-[(1-Methylethyl)oxy]l-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

To a stirred suspension of8-bromo-2-[(1-methylethyl)oxy]-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(690 mg, 1.937 mmol) in methanol (15 ml) was added sodium methoxide (30%wt/v solution in methanol, 2.4 ml) and the reaction mixture heated at 5°C. for 2 hours. The reaction mixture was then heated to 70° C. andstirred for 2.5 hours. The solvent was evaporated and the residuepartioned between saturated aqueous ammonium chloride solution (15 ml)and ethyl acetate (20 mL). The layers were separated, the aqueous phasewas extracted with additional ethyl acetate (2×10 mL) and the organicextracts were combined, dried by passage through a hydrophobic frit andevaporated to give the title compound as a yellow solid (573 mg).

LCMS (System B): t_(RET)=1.92 min; MH⁺=308

Intermediate 53: 2-[(1-Methylethyl)oxy]l-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate

Trifluoroacetic acid (1 ml, 12.98 mmol) was added to a stirred solutionof2-[(1-methylethyl)oxy]-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(568 mg, 1.848 mmol) in methanol (10 ml) and the mixture was stirred atroom temperature overnight. More trifluoroacetic acid (0.2 ml) was addedand the reaction mixture stirred at room temperature for a further 1.5hours and then evaporated in vacuo. The solid residue was trituratedwith ethyl acetate, collected by filtration, washed with ethyl acetateand dried in vacuo overnight to give the title compound as a white solid(405 mg).

LCMS (System B): t_(RET)=1.02 min; MH⁺=224

Intermediate 54:9-(5-Chloropentyl)-2-[(1-methylethyl)oxy]l-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 44 from2-[(1-methylethyl)oxy]-8-(methyloxy)-1H-purin-6-amine trifluoroacetateand 1-bromo-5-chloropentane.

LCMS (System A): t_(RET)=0.93 min; MH⁺=328/330

Intermediate 55:2-(Cyclobutyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Sodium t-butoxide (3.31 g, 34.2 mmol) was added portionwise tocyclobutanol (10 ml) at room temperature. The mixture became very thickand was heated to 50° C.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (2 g, 8.43 mmol)was added followed by 1,2-dimethoxyethane (3 ml) and the mixture stirredat 50° C. for 90 mins. and then cooled and partitioned between ethylacetate (50 ml) and water (50 ml). A precipitate that failed to dissolvein either phase was removed by filtration. The organic phase wasseparated, washed with saturated brine, dried over anhydrous magnesiumsulphate, filtered and evaporated to give a cream foam. This materialwas dissolved in dichloromethane and purified on an aminopropyl (NH₂)cartridge (110 g) using a 0-100% ethyl acetate in cyclohexane gradientfollowed by a 0-20% methanol (+1% triethylamine) gradient over 40 mins.The appropriate fractions were combined and evaporated in vacuo to givethe title compound as an off-white solid (0.655 g).

LCMS (System B): t_(RET)=1.98 min; MH⁺=290

Intermediate 56:8-Bromo-2-(cyclobutyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (1.152 g, 6.47 mmol) was added to a stirred solutionof 2-(cyclobutyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.248 g, 4.31 mmol) in chloroform (15 ml) at 0° C. The mixture waswarmed to room temperature and left overnight when water (15 ml) wasadded and the phases separated. The aqueous layer was extracted withdichloromethane and the organic extracts were combined, washed withbrine, dried over anhydrous magnesium sulphate and evaporated to givethe title compound as an orange foam (1.79 g).

LCMS (System D): t_(RET)=2.72 min; MH⁺=368/370

Intermediate 57:2-(Cyclobutyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

8-Bromo-2-(cyclobutyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.79 g, 4.86 mmol) was dissolved in anhydrous methanol (25 ml) and 25%sodium methoxide in methanol (2.274 ml, 9.72 mmol) was added undernitrogen. The mixture was heated at 67° C. for 24 hours and then cooledto room temperature. Ethyl acetate and water were added and the layersseparated. The aqueous layer was extracted twice more with ethylacetate, and the organic extracts were combined, washed with brine,dried over anhydrous magnesium sulfate, and evaporated to give the titlecompound as a cream foam (1.27 g).

LCMS (System D): t_(RET)=2.53 min; MH⁺=320

Intermediate 58: 2-(Cyclobutyloxy)-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate

Trifluoroacetic acid (3 ml, 38.9 mmol) was added to a solution of2-(cyclobutyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(1.27 g, 3.98 mmol) in methanol (50 ml) and the mixture stirred at 20°C. under an atmosphere of nitrogen for 21 hours. The solvent was removedin vacuo, and the residual solid was triturated with 1,1-dimethylethylmethyl ether and then collected by filtration and dried in vacuo to givethe title compound as a cream solid (1.0922 g).

LCMS (System D): t_(RET)=1.17 min; MH⁺=236

Intermediate 59:9-(4-Chlorobutyl)-2-(cyclobutyloxy)-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 44 from2-(cyclobutyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and1-bromo-4-chlorobutane.

LCMS (System D): t_(RET)=2.76 min; MH⁺=326/328

Intermediate 60:2-(Cyclopentyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Cyclopentanol (25 ml, 275 mmol) was added to sodium tert-butoxide (4.05g, 42.2 mmol) to give a thick suspension which was diluted with1,2-dimethoxyethane (35 ml) and heated to 50° C.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (2.5 g, 10.54mmol) was added to the resulting solution which was then stirred undernitrogen at 50° C. for 20 hours. The mixture was cooled and water andethyl acetate were added. The layers separated and the aqueous layerwashed again with ethyl acetate. The organic extracts were combined,washed with brine, dried over anhydrous magnesium sulphate andconcentrated under reduced pressure at 40° C. The residue was loaded incyclohexane (50 ml) onto 330 g silica cartridge and eluted firstly witha 0-100% ethyl acetate in cyclohexane gradient over 10 column volumesand then with a 0-30% methanol in ethyl acetate gradient.Product-containing fractions were combined and evaporated to give thetitle compound as a white foam (2.51 g).

LCMS (System D): t_(RET)=2.51 min; MH⁺=304

Intermediate 61:8-Bromo-2-(cyclopentyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Prepared similarly to Intermediate 56 from2-(cyclopentyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.88 min; MH⁺=382/384

Intermediate 62:2-(Cyclopentyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Prepared similarly to Intermediate 57 from8-bromo-2-(cyclopentyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System C): t_(RET)=1.1 min; MH⁺=334

Intermediate 63: 2-(Cyclopentyloxy)-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate

Prepared similarly to Intermediate 58 from2-(cyclopentyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.27 min; MH⁺=250

Intermediate 64:9-(4-Chlorobutyl)-2-(cyclopentyloxy)-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 44 from2-(cyclopentyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and1-bromo-4-chlorobutane.

LCMS (System D): t_(RET)=2.90 min; MH⁺=340/342

Intermediate 65:2-(Cyclohexyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Sodium tert-butoxide (3.29 g, 34.2 mmol) was added portionwise tocyclohexanol (15 ml) at room temperature. The mixture became very thickand more cyclohexanol (10 ml) was added and the mixture heated to 50° C.2-Fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (2 g, 8.43 mmol)was added and the mixture heated at 50° C. for 1 hour and then warmed to60° C. and heated for a further 2 hours at which point LCMS showedcomplete reaction. The mixture was cooled to room temperature andpartitioned between ethyl acetate (150 ml) and water (150 ml). Theorganic phase was separated, washed with saturated brine, dried overanhydrous magnesium sulphate, filtered and evaporated on a water bath at60° C. The residue was dissolved in dichloromethane and purified on a 70g aminopropyl (NH₂) cartridge using a 0-100% ethyl acetate incyclohexane gradient followed by a 0-20% methanol (+1% triethylamine)gradient over 30 mins. Some product-containing fractions werecontaminated with cyclohexanol and these were re-purified on a 70 gsilica cartridge using a 0-100% ethyl acetate-cyclohexane gradient over40 mins. Product-containing fractions from the two purifications werecombined and evaporated in vacuo to give the title compound as a paleyellow foam (1.59 g).

LCMS (System D): t_(RET)=2.65 min; MH⁺=318

Intermediate 66:8-Bromo-2-(cyclohexyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

N-Bromosuccinimide (0.214 g, 1.2 mmol) was added to a stirred solutionof 2-(cyclohexyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine(0.254 g, 0.80 mmol) in chloroform (5 ml) at 0° C. The resultant mixturewas stirred at 0° C. for 1.5 hours and then warmed to room temperatureand stirred for a further 2 hours. Water (5 ml) was added and the phasesseparated using a hydrophobic frit. The organic phase was evaporated andthe residue dissolved in dichloromethane and purified on a 70 gaminopropyl (NH₂) cartridge eluting with a 0-100% ethyl acetate incyclohexane gradient over 40 mins. The appropriate fractions werecombined and evaporated in vacuo to give the title compound as a whitesolid (0.252 g).

LCMS (System B): t_(RET)=2.83 min; MH⁺=396/398

Intermediate 67:2-(Cyclohexyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine

Prepared similarly to Intermediate 57 from8-bromo-2-(cyclohexyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System D): t_(RE)T=2.86 min; MH⁺=348

Intermediate 68: 2-(Cyclohexyloxy)-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate

Prepared similarly to Intermediate 58 from2-(cyclohexyloxy)-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.43 min; MH⁺=264

Intermediate 69:9-(4-Chlorobutyl)-2-(cyclohexyloxy)-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 44 from2-(cyclohexyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate and1-bromo-4-chlorobutane.

LCMS (System D): t_(RET)=3.05 min; MH⁺=354/356

Intermediate 70:N²-[(1R)-1-Methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

A crude sample of (2R)-2-pentanamine containing dichloromethane (11.12 gcontaining ca 3.1 g, 35.6 mmol of amine) was added to a suspension of2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine (5.00 g, 21.08mmol) in ethylene glycol (50 ml). The mixture was heated at 110° C. for20 hours and then cooled to room temperature and partitioned betweenwater (200 ml) and ethyl acetate (200 ml). The organic phase wasseparated, washed with saturated brine, dried over anhydrous magnesiumsulphate, filtered and evaporated. The residue was dissolved indichloromethane and purified on a 110 g aminopropyl (NH₂) cartridgeusing a 0-100% ethyl acetate-cyclohexane gradient over 40 mins. Theappropriate fractions were combined and evaporated in vacuo and theresidue triturated with diethyl ether and some insoluble startingmaterial removed by filtration. Evaporation of the ether filtrateafforded the title compound as an off-white foam (2.34 g).

LCMS (System D): t_(RET)=2.63 min; MH⁺=305

Intermediate 71:8-Bromo-N²-[(1R)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

N-Bromosuccinimide (2.08 g, 11.69 mmol) was added portionwise to astirred solution ofN²-[(1R)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine(2.27 g, 7.46 mmol) in chloroform (30 ml) at 0° C. under at atmosphereof nitrogen. The reaction mixture was allowed to stir for 1.5 hours whenchloroform (20 ml) and water (50 ml) were added. After mixing the layerswere separated using a hydrophobic frit, the aqueous layer was washedwith an additional portion of chloroform and the combined organicextracts were evaporated. The residue was dissolved in dichloromethaneand purified on a 110 g aminopropyl (NH₂) cartridge using a 0-100% ethylacetate in cyclohexane gradient over 40 mins. The appropriate fractionswere combined and evaporated in vacuo to give the title compound as anoff-white foam (0.846 g).

LCMS (System D): t_(RET)=3.05 min; MH⁺=383/385

Intermediate 72:N²-[(1R)-1-Methylbutyl]-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

A solution of sodium methoxide in methanol (0.5M, 9 ml, 4.5 mmol) wasadded to a solution of8-bromo-N²-[(1R)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine(0.844 g, 2.20 mmol) in methanol (12 ml) and the resulting solutionheated under reflux for 23.5 hours. More sodium methoxide in methanol(0.5M, 4.5 ml) was then added and refluxing continued for a further 4hours. More sodium methoxide in methanol (0.5M, 4.5 ml) was again addedand refluxing continued for a further 16.5 hours when LCMS indicatedreaction to be complete. The reaction mixture was cooled to roomtemperature, evaporated and the residue partitioned between ethylacetate (75 ml) and water (75 ml). The aqueous phase was re-extractedwith ethyl acetate (75 ml) and the combined organic phases were washedwith saturated brine, dried over anhydrous magnesium sulphate, filteredand evaporated. The residue was dissolved in dichloromethane andpurified on a 100 g aminopropyl (NH₂) cartridge using a 0-100% ethylacetate in cyclohexane gradient followed by a 0-20% methanol (+1%triethylamine) gradient over 15 mins. Product-containing fractions werecombined and evaporated in vacuo to give the title compound as a whitefoam (0.614 g).

LCMS (System D): t_(RET)=2.83 min; MH⁺=335

Intermediate 73:N²-[(1R)-1-Methylbutyl]-8-(methyloxy)-3H-purine-2,6-diaminetrifluoroacetate

Trifluoroacetic acid (1 ml, 1.48 g, 7.08 mmol) was added to a stirredsolution ofN₂-[(1R)-1-methylbutyl]-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine(0.613 g, 1.833 mmol) in methanol (10 ml). The resultant mixture wasstirred for 66 hours under an atmosphere of nitrogen and then evaporatedto give the title compound as an off-white solid (0.690 g).

LCMS (System D): t_(RET)=1.89 min; MH⁺=251

Intermediate 74:9-(4-Chlorobutyl)-N²-[(1R)-1-methylbutyl]-8-(methyloxy)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 44 fromN²-[(1R)-1-methylbutyl]-8-(methyloxy)-3H-purine-2,6-diaminetrifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): t_(RE)T=3.02 min; MH⁺=341/343 Intermediate 75:N²-[(1S)-1-Methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 70 from2-fluoro-9-(tetrahydro-2H-pyran-2-yl)-9H-purin-6-amine and(2S)-2-pentanamine.

LCMS (System D): t_(RET)=2.63 min; MH⁺=305

Intermediate 76:8-Bromo-N²-[(1S)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 71 fromN²-[(1S)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=3.05 min; MH⁺=383/385

Intermediate 77:N²-[(1S)-1-Methylbutyl]-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine

A solution of sodium methoxide in methanol (0.5M, 13 ml, 6.5 mmol) wasadded to a solution of 8-bromo-N²-[(1S)-1-methylbutyl]-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine(1.26 g, 3.29 mmol) in methanol (10 ml) and the resulting solutionheated under reflux for 4 hours. More sodium methoxide in methanol(0.5M, 12 ml, 6 mmol) was then added and refluxing continued for afurther 18 hours. The mixture was cooled and evaporated and the residuepartitioned between ethyl acetate (75 ml) and water (75 ml). The aqueousphase was re-extracted with ethyl acetate (75 ml) and the combinedorganic phases were washed with saturated brine, dried over anhydrousmagnesium sulphate and evaporated. The residue was dissolved indichloromethane and purified on a 100 g aminopropyl (NH₂) cartridgeusing a 0-100% ethyl acetate in cyclohexane gradient followed by a 0-20%methanol (+1% triethylamine) gradient over 15 mins. Theproduct-containing fractions were combined and evaporated in vacuo togive the title compound as a white foam (0.848 g).

LCMS (System D): t_(RET)=2.83 min; MH⁺=335

Intermediate 78: N²-[(1S)-1-Methylbutyl]-8-(methyloxy)-3H-purine-2,6-diamine trifluoroacetate

Prepared similarly to Intermediate 73 fromN²-[(1S)-1-methylbutyl]-8-(methyloxy)-9-(tetrahydro-2H-pyran-2-yl)-9H-purine-2,6-diamine.

LCMS (System D): t_(RET)=1.89 min; MH⁺=251

Intermediate 79: 9-(4-Chlorobutyl)-N²-[(1S)-1-methylbutyl]-8-(methyloxy)-9H-purine-2,6-diamine

Prepared similarly to Intermediate 44 fromN²-[(1S)-1-methylbutyl]-8-(methyloxy)-3H-purine-2,6-diaminetrifluoroacetate and 1-bromo-4-chlorobutane.

LCMS (System D): t_(RET)=3.02 min; MH⁺=341/343

Intermediate 80: 9-(3-Chloropropyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine

Prepared similarly to Intermediate 44 from 2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine trifluoroacetateand 1-bromo-3-chloropropane.

LCMS (System D): t_(RET)=2.90 min; MH⁺=328/330

Intermediate 81: 9-(5-Chloropentyl)-8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9H-purin-6-amine

Prepared similarly to Intermediate 14 from8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-1H-purin-6-aminetrifluoroacetate and 1-bromo-5-chloropentane.

LCMS (System A): t_(RET)=1.00 min; MH⁺=342/344

Intermediate 82: 8-(Methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine

Prepared similarly to Intermediate 38 from9-(5-chloropentyl)-8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9H-purin-6-amine and piperidine but withpurification on silica using a 0-25% methanol in dichloromethanegradient.

LCMS (System A): t_(RET)=0.61 min; MH⁺=391

Intermediate 83:2-(Butyloxy)-8-(methyloxy)-9-[3-(1-piperidinyl)propyl]-9H-purin-6-amine,Formic Acid Salt

Prepared similarly to Intermediate 20 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1,3-dibromopropane and piperidine but with sequential purifications byMDAP using Method A followed by Method D.

LCMS (System B): t_(RET)=1.16 min; MH⁺=363

Example 1:6-Amino-9-[3-(1-azetidinyl)propyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one

9-[3-(1-Azetidinyl)propyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine(13 mg, 0.039 mmol) was dissolved in methanol (3 ml) and 4M hydrogenchloride in 1,4-dioxane (0.243 ml, 0.972 mmol) was added and the mixturestirred at room temperature for 18 hours. The solvent was removed invacuo and the residue was dissolved in methanol and loaded onto anaminopropyl SPE cartridge (2 g). The cartridge was eluted with methanoland the solvent removed to give the title compound as a white solid (13mg).

LCMS (System B): t_(RET)=1.12 min; MH⁺=321

Example 2:6-Amino-2-(butyloxy)-9-[3-(1-pyrrolidinyl)propyl]-7,9-dihydro-8H-purin-8-one

2-(Butyloxy)-8-(methyloxy)-9-[3-(1-pyrrolidinyl)propyl]-9H-purin-6-amine(49 mg, 0.141 mmol) was dissolved in methanol (5 ml) and 4M hydrogenchloride in 1,4-dioxane (0.879 ml, 3.52 mmol) was added and the mixturestirred at room temperature for 5 hours. The solvent was removed invacuo to give a cream solid which was dissolved in methanol and loadedonto an aminopropyl SPE cartridge (2 g) and eluted with methanol. Thesolvent was evaporated to give the title compound as a white solid (43mg).

LCMS (System C): t_(RET)=0.70 min; MH⁺=335

Example 3:6-Amino-2-(butyloxy)-9-[3-(hexahydro-1H-azepin-1-yl)propyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-9-[3-(hexahydro-1H-azepin-1-yl)propyl]-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.33 min; MH⁺=363

Example 4:6-Amino-9-[4-(1-azetidinyl)butyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from9-[4-(1-azetidinyl)butyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.16 min; MH⁺=335

Example 5:6-Amino-2-(butyloxy)-9-[4-(1-pyrrolidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[4-(1-pyrrolidinyl)butyl]-9H-purin-6-amineformic acid salt.

LCMS (System B): t_(RET)=1.23 min; MH⁺=349

Example 6:6-Amino-2-(butyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purin-6-amineformic acid salt.

LCMS (System B): t_(RET)=1.29 min; MH⁺=363

Example 7:6-Amino-2-(butyloxy)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.37 min; MH⁺=377

Example 8:6-Amino-9-[5-(1-azetidinyl)pentyl]-2-(butyloxy)-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from9-[5-(1-azetidinyl)pentyl]-2-(butyloxy)-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.25 min; MH⁺=349

Example 9:6-Amino-2-(butyloxy)-9-[5-(1-pyrrolidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[5-(1-pyrrolidinyl)pentyl]-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.28 min; MH⁺=363

Example 10:6-Amino-2-(butyloxy)-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.35 min; MH⁺=377

Example 11:6-Amino-2-(butyloxy)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-7,9-dihydro-8H-purin-8-one

Method A

Prepared similarly to Example 2 from2-(butyloxy)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.55 min; MH⁺=391

Method B

Prepared similarly to Example 19 from2-(butyloxy)-8-(methyloxy)-1H-purin-6-amine trifluoroacetate,1-bromo-5-chloropentane and hexahydro-1H-azepine.

LCMS (System B): t_(RET)=1.54 min; MH⁺=391

Example 12:6-Amino-2-(butyloxy)-9-[5-(hexahydro-1(2H)-azocinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-9-[5-(hexahydro-1(2H)-azocinyl)pentyl]-8-(methyloxy)-9H-purin-6-amine.

LCMS (System D): t_(RET)=3.17 min; MH⁺=405

Example 13:6-Amino-2-(butyloxy)-9-[6-(1-pyrrolidinyl)hexyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[6-(1-pyrrolidinyl)hexyl]-9H-purin-6-amine.

LCMS (System D): t_(RE)T=2.47 min; MH⁺=377

Example 14:6-Amino-2-(butyloxy)-9-[6-(1-piperidinyl)hexyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[6-(1-piperidinyl)hexyl]-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.68 min; MH⁺=391

Example 15:6-Amino-2-(butyloxy)-9-[6-(hexahydro-1H-azepin-1-yl)hexyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-9-[6-(hexahydro-1H-azepin-1-yl)hexyl]-8-(methyloxy)-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.76 min; MH⁺=405

Example 16:6-Amino-2-(butylamino)-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one

A mixture ofN²-butyl-9-(3-chloropropyl)-8-(methyloxy)-9H-purine-2,6-diamine (250 mg,0.8 mmole), piperidine (340 mg, 4 mmole) and sodium iodide (360 mg, 2.4mmole) in THF (8 ml) was heated under reflux for 48 hours. The solventwas evaporated and the residue purified by preparative TLC thendissolved in methanol (5 ml). Hydrogen chloride in methanol (0.5 ml) wasadded and the mixture stirred at room temperature for 16 hours. Thesolvent was then evaporated and the pH of the residue was adjusted to7-8 by addition of sodium bicarbonate solution. The product wasextracted into ethyl acetate and the extract was evaporated and theresidue purified by preparative HPLC to give the title compound (16 mg).

LCMS (System A): t_(RET)=0.55 min; MH⁺=348

Example 17:6-Amino-2-(butylamino)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 fromN²-butyl-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purine-2,6-diamine.

LCMS (System B): t_(RET)=0.96 min; MH⁺=362

Example 18:6-Amino-2-(butylamino)-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 fromN²-butyl-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-8-(methyloxy)-9H-purine-2,6-diamine.

LCMS (System B): t_(RET)=1.12 min; MH⁺=376

Example 19:6-Amino-2-(butylamino)-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-7,9-dihydro-8H-purin-8-one

N²-Butyl-8-(methyloxy)-3H-purine-2,6-diamine trifluoroacetate (192 mg,0.547 mmol) and potassium carbonate (189 mg, 1.368 mmol) were suspendedin DMF (3 ml) and heated to 60° C. for 1 hour. The reaction mixture wascooled to room temperature and 1-bromo-5-chloropentane (0.072 ml, 0.547mmol) was added and the reaction stirred for a further 18 hours.Hexahydro-1H-azepine (54.2 mg, 0.547 mmol) and triethylamine (0.076 ml,0.547 mmol) were added and the reaction mixture heated to 70° C. for 24hours. LCMS showed a major peak with MH⁺404 consistent with theformation ofN²-butyl-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-8-(methyloxy)-9H-purine-2,6-diamine.The solvent was removed in vacuo and the residue partitioned between DCM(2 ml) and water (2 ml). The aqueous was extracted again with DCM (2 ml)and the combined organic extracts were concentrated and the residue wasdissolved in 1:1 MeOH:DMSO (2 ml) and purified by MDAP (Method C).Evaporation of the product containing fractions gave a residual TFA saltwhich LCMS indicated had undergone hydrolysis of the 8-methoxy group,presumably on concentration in the presence of TFA. This crude materialwas dissolved once more in 1:1 MeOH:DMSO (2 ml) and re-purified by MDAP(Method A). Product-containing fractions were evaporated under a streamof nitrogen to give the title compound as a white solid (39 mg).

LCMS (System B): t_(RET)=1.18 min; MH⁺=390

Example 20: 6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 19 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate, 1-bromo-4-chlorobutane and piperidine.

LCMS (System B): t_(RET)=1.38 min; MH⁺=377

Example 21:6-Amino-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-2-{[(1S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from9-[4-(hexahydro-1H-azepin-1-yl)butyl]-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.48 min; MH⁺=391

Example 22: 6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-{[(1S)-1-Methylbutyl]oxy}-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amineas follows:

A solution of hydrogen chloride in dioxane (4M, 0.71 ml) was added to asolution of 2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine(0.046 g, 0.126 mmol) in methanol (3 ml). The resultant mixture wasallowed to stand overnight at room temperature and then blown down undernitrogen. The residue was dissolved in methanol and loaded onto a 2 gaminopropyl SPE cartridge (pre-conditioned with methanol), eluted withmethanol and the resultant solution blown down under nitrogen to givethe title compound as a yellow solid (40.97 mg).

LCMS (System D): t_(RET)=2.70 min; MH⁺=391

A similarly prepared sample (1.7 g) was recrystallised from ethylacetate (ca 50 ml). The crystals were collected, washed with ice-coldethyl acetate (15 ml) and dried in vacuo at 50° C. for 3 hours to givethe title compound as a cream crystalline solid (1.33 g).

-   Melting point onset (DSC): 207.4° C. (see FIG. 2)-   XRPD: (see FIG. 1 and Table 1)

Example 23: 6-Amino-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-2-{[(1S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 19 from2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-1H-purin-6-aminetrifluoroacetate, 1-bromo-5-chloropentane and hexahydro-1H-azepine.

LCMS (System B): t_(RET)=1.54 min; MH⁺=405

Example 24:6-Amino-2-{[(1S)-1-methylpropyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9H-purin-6-amineand piperidine.

LCMS (System D): t_(RET)=2.27 min; MH⁺=363

A sample of the intermediate 8-methoxy derivative8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9-[4-(1-piperidinyl)butyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=2.56 min; MH⁺=377

Example 25:6-Amino-2-{[(1S)-1-methylpentyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-8-(methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-9H-purin-6-amine and piperidine.

LCMS (System D): t_(RET)=2.72 min; MH⁺=391

A sample of the intermediate 8-methoxy derivative8-(methyloxy)-2-{[(1S)-1-methylpentyl]oxy}-9-[4-(1-piperidinyl)butyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=3.01 min; MH⁺=405

Example 26:6-Amino-2-[(1-methylethyl)oxy]l-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(5-chloropentyl)-2-[(1-methylethyl)oxy]-8-(methyloxy)-9H-purin-6-amineand piperidine.

LCMS (System D): t_(RET)=2.18 min; MH⁺=363

A sample of the intermediate 8-methoxy derivative2-[(1-methylethyl)oxy]-8-(methyloxy)-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=2.43 min; MH⁺=377

Example 27:6-Amino-2-(cyclobutyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-2-(cyclobutyloxy)-8-(methyloxy)-9H-purin-6-amine andpiperidine.

LCMS (System D): t_(RET)=2.24 min; MH⁺=361

A sample of the intermediate 8-methoxy derivative2-(cyclobutyloxy)-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=2.49 min; MH⁺=375

Example 28:6-Amino-2-(cyclopentyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-2-(cyclopentyloxy)-8-(methyloxy)-9H-purin-6-amine andpiperidine.

LCMS (System D): t_(RET)=2.38 min; MH⁺=375

A sample of the intermediate 8-methoxy derivative2-(cyclopentyloxy)-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=2.64 min; MH⁺=389

Example 29:6-Amino-2-(cyclohexyloxy)-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Sodium iodide (0.006 g, 0.04 mmol) was added to a stirred mixture of9-(4-chlorobutyl)-2-(cyclohexyloxy)-8-(methyloxy)-9H-purin-6-amine(0.103 g, 0.303 mmol), N,N-diisopropylethylamine (0.105 ml, 0.079 g,0.609 mmol) and piperidine (0.120 ml, 0.103 g, 1.215 mmol) in DMF (1.5ml). The resultant mixture was heated at 80° C. for 20 hours when LCMSshowed the formation of two products, one corresponding to displacementof the chloride by piperidine and the second corresponding toconcomitant hydrolysis of the 8-methoxy moiety. The reaction mixture waspartitioned between dichloromethane (6 ml) and water (6 ml) and thephases separated using a hydrophobic frit. The solvent was removed fromthe organic phase under a stream of nitrogen in a blow-down unit and theresidue was dissolved in 1:1 MeOH:DMSO (2 ml) and separated by massdirected autopreparation (Method A) to afford the title compound as awhite solid (16.6 mg).

LCMS (System D): t_(RET)=2.53 min; MH⁺=389

The intermediate2-(cyclohexyloxy)-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purin-6-aminewas also isolated as a colourless solid (55.2 mg).

LCMS (System D): t_(RET)=2.80 min; MH⁺=403

Example 30:6-Amino-2-{[(1R)-1-methylbutyl]amino}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-N²-[(1R)-1-methylbutyl]-8-(methyloxy)-9H-purine-2,6-diamineand piperidine.

LCMS (System D): t_(RET)=2.47 min; MH⁺=376

A sample of the intermediate 8-methoxy derivativeN²-[(1R)-1-methylbutyl]-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purine-2,6-diaminewas also isolated.

LCMS (System D): t_(RET)=2.76 min; MH⁺=390

Example 31:6-Amino-2-{[(1S)-1-methylbutyl]amino}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from9-(4-chlorobutyl)-N²-[(1S)-1-methylbutyl]-8-(methyloxy)-9H-purine-2,6-diamineand piperidine.

LCMS (System D): t_(RET)=2.47 min; MH⁺=376

A sample of the intermediate 8-methoxy derivativeN²-[(1S)-1-methylbutyl]-8-(methyloxy)-9-[4-(1-piperidinyl)butyl]-9H-purine-2,6-diaminewas also isolated.

LCMS (System D): t_(RET)=2.76 min; MH⁺=390

Example 32: 6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 29 from 9-(3-chloropropyl)-2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9H-purin-6-amine and piperidine.

LCMS (System D): t_(RET)=2.52 min; MH⁺=363

A sample of the intermediate 8-methoxy derivative2-{[(1S)-1-methylbutyl]oxy}-8-(methyloxy)-9-[3-(1-piperidinyl)propyl]-9H-purin-6-aminewas also isolated.

LCMS (System D): t_(RET)=2.87 min; MH⁺=377

Example 33: 6-Amino-2-{[(1S)-1-methylpropyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from 8-(methyloxy)-2-{[(1S)-1-methylpropyl]oxy}-9-[5-(1-piperidinyl)pentyl]-9H-purin-6-amine.

LCMS (System D): t_(RET)=2.39 min; MH⁺=377

Example 34:6-Amino-2-(butyloxy)-9-[3-(1-piperidinyl)propyl]-7,9-dihydro-8H-purin-8-one

Prepared similarly to Example 1 from2-(butyloxy)-8-(methyloxy)-9-[3-(1-piperidinyl)propyl]-9H-purin-6-amine.

LCMS (System B): t_(RET)=1.23 min; MH⁺=349

Polymorphism

X-ray powder diffraction (XRPD) and differential scanning calorimetry(DSC) were performed on 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneaccording to the following methods.

XRPD

XRPD data were acquired on a PANalytical X'Pert Pro powderdiffractometer, equipped with an X'Celerator detector. The acquisitionconditions were: radiation: Cu Kα, generator tension: 40 kV, generatorcurrent: 45 mA, start angle: 2.0° 2θ, end angle: 40.0° 2θ, step size:0.0167° 2θ. The time per step was 31.750 s. The sample was prepared bymounting a few milligrams of sample on a Si wafer (zero background)plate, resulting in a thin layer of powder.

Characteristic peak positions and calculated d-spacings are summarisedin Table 1. These were calculated from the raw data using Highscoresoftware. The experimental error in the peak positions is approximately±0.1° 2θ. Relative peak intensities will vary due to preferredorientation.

TABLE 1 Characteristic XRPD Peak Positions for Solid-state Form 1 of6-Amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one Form 1 2θ/°d-spacing/Å 5.0 17.6 10.0 8.8 12.7 7.0 13.5 6.5 13.8 6.4 16.6 5.3 18.94.7 20.0 4.4 22.2 4.0 23.3 3.8 24.2 3.7 26.1 3.4

A representative XRPD diffractogram of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis shown in FIG. 1.

DSC

The DSC thermogram was obtained using a TA Instruments calorimeter. Thesample was weighed into an aluminium pan, a pan lid placed on top andlightly crimped without sealing the pan. The experiment was conductedusing a heating rate of 10° C. min⁻¹.

A representative DSC thermogram of 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-oneis shown in FIG. 2.

Biological Data

Compounds of the invention were tested for in vitro biological activityin accordance with the following assays, or similar assays:

Assay for the Induction of Interferon-α Using Cryopreserved HumanPeripheral Blood Mononuclear Cells (PBMCs) Compound Preparation

Compounds were dissolved in DMSO. Serial 2-fold dilutions with DMSO wereprepared and 0.25 μl dispensed into 384-well clear Greiner polypropyleneplates.

Preparation of PBMCs

Blood samples of up to 200 ml were obtained from healthy human donors.Whole blood in 25 ml volumes was overlaid onto 15 ml Ficoll® gradientsin Leucosep® tubes, and centrifuged at 1000 g for 20 min. Cells in theband at the plasma/Histopaque® interface were carefully removed andwashed twice with PBS (centrifuged at 400 g for 5 min to harvest). Thefinal pellet was resuspended in freezing medium (90% Heat-inactivatedserum, 10% DMSO) to a cell concentration of 4×10⁷ cells/ml. Theresuspended cells were then cryopreserved (frozen) using a ratecontrolled freezer, and stored at −140° C. for up to 4 months.

Incubation and Assay for Interferon-α

Immediately prior to assay, vials of cryopreserved (frozen) PBMCs werethawed rapidly in a water bath at 37° C. A 1:10 dilution of the cells intrypan blue was prepared and counted. The PBMCs were then diluted ingrowth media [RPMI 1640 containing 10% fetal calf serum (invitrogen),Penicillin+Streptavidin (Gibco cat. #25030-024, 1:50), L-Glutamine 2 mM,and 1000 units/ml recombinant human IFN-gamma (Preprotech catalogue#300-02)] to a density of 1×10⁶ cells/ml, and 50 ul/well dispensed to384-well clear Greiner polypropylene plates containing 0.25l DMSO ortest compound in 0.25l DMSO. Top final concentration of compound wastypically 50 uM or 5 uM (to obtain curve fit for highly activecompounds). Plates were incubated for 24 h at 37° C. in 5% CO₂.

A multi-isoform immunoassay was used to quantify IFN-α in PBMCsupernatants. Rabbit polyclonal antibody against human IFN-α (cataloguenumber 31101, Stratech Scientific) was diluted 1:10000 in assay buffer(RPMI 1640 containing 10% fetal calf serum, Invitrogen) and 20 μl wasadded to each well of an MSD® (Meso-Scale® Discovery) single small-spot384-well GAR (goat anti-rabbit antibody coated) plate. The plate wasincubated for 1 h at room temperature with vigorous shaking. Followingthree washes with PBS, 20 μl of cell supernatant were added to each wellof the plate. The plate was then incubated for 1 h at room temperaturewith vigorous shaking. A pair of monoclonal antibodies to IFN-α(catalogue numbers 21100 and 21112, Stratech Scientific) were labelledwith sulfo-TAG (MSD®), diluted 1:1000 in assay buffer and 20 μl added toeach well of the plate. The plate was further incubated for 1 h at roomtemperature with vigorous shaking. Following three washes with PBS, 30μl of ×2 T buffer (MSD®) was added to each well and the plate was readon an MSD® Sector 6000 plate reader.

Data were normalised to internal plate controls of 1 uM resiquimod(n=16) and DMSO (n=16). pEC50 values were derived by 4-parameter curvefit with IRLS in ActivityBase, from 11-point, two-fold serial dilutionof test compounds.

Results

Examples 1 to 34 had a mean pEC₅₀ of >5.5.

Assay for the Induction of Interferon-α and TNF-α Using Fresh HumanPeripheral Blood Mononuclear Cells (PBMCs)

Compound preparation Compounds were dissolved and serially diluted inDMSO to give 100× the required concentration range using a Biomek® 2000.1 ul of test compound was transferred into 96-well tissue culture platesusing a Biomek® FX. Each compound was assayed in duplicate for eachdonor. Each plate contained a dilution series of the TLR7/8 agonistresiquimod as standard and Column 11 contained 1 μl of 200 μM resiquimod(giving a 2 μM final concentration, used to define the approximatemaximal response to resiquimod).

Preparation of PBMCs

Blood samples from two human donors were collected into sodium heparin(10 U/ml). 25 ml volumes of whole blood were overlaid onto 15 mlsHistopaque® in LEUCOSEP™ tubes which were centrifuged at 800 g for 20min and the band at the plasma/histopaque interface carefully removed.The collected cells were centrifuged at 2500 rpm for 10 min and thepellet resuspended in 10 ml of media (RPMI 1640 (Low endotoxin)supplemented with 10% v/v foetal calf serum (FCS, low endotoxin) 100U/ml penicillin G, 100 μg/ml streptomycin, 10 mM L-glutamine and 1×non-essential amino acids). A 1:20 dilution of the cells was preparedusing trypan blue & the cells counted using a haemocytometer. The PBMCswere diluted to give a final concentration of 2×10⁶/ml and 100 ul ofthis cells suspension was added to wells containing 1 μl of diluted testcompound.

Incubation and Assays for Interferon-α and TNF-α

The cell preparations were incubated for 24 hr (37° C., 95% air, 5% CO₂)after which a sample of the supernatant was removed using the Biomek® FXand assayed for both IFN-α and TNF-α using the MSD® (MesoscaleDiscovery) electrochemiluminescence assay platform. The IFN-α assay wascarried out similarly to that described above. The TNF-α assay wascarried out as per kit instructions (Cat No K111BHB).

Cytokine released was expressed as a percentage of the 2 μM resiquimodcontrol (column 11). This percentage was plotted against compoundconcentration and the pEC50 for the response determined by non-linearleast squares curve fitting. For the IFN-α responses generally a 4parameter logistic model was selected. For the TNF responses where aclear maximum response was obtained (i.e. a well defined plateau in theresponse was observed) then a 4 parameter model was generally used. Ifthe upper asymptote of the curve wasn't well defined then the curvefitting was generally constrained to a maximal response of 100% (i.e. tothe response to 2 μM resiquimod) or to the response of the highestconcentration tested if this was greater than the resiquimod response.Some curves were bell shaped for one or both cytokines and the cytokinedata on the down slope of the bell shaped response (i.e. concentrationsabove those giving the maximal response) were generally excluded fromthe fit, usually with the exception of the concentration immediatelyabove the peak response. Curve fitting thus concentrated on the up slopeof the dose response curve.

Results

Examples 5 and 9 showed mean pEC₅₀s for induction of IFN-α and TNF-αof >7.5 and <5.5 respectively. Examples 6, 7, 10 to 12, 14, and 18showed mean pEC₅₀s for induction of IFN-α and TNF-α of ≥8 and <6respectively. Examples 13, 15 and 20 to 23 showed mean pEC₅₀s forinduction of IFN-α and TNF-α of ≥9 and ≤6.5 respectively.

Allergen-Driven Cytokine Assay Using Fresh Human Peripheral BloodMononuclear Cells (PBMCs) from Atopic Volunteers

An assay based on co-culture of atopic human donor derived peripheralblood mononuclear cells (PBMCs) with allergen and test compounds wasdeveloped. After 5-6 days culture, cell supernatants were assayed for arange of cytokines.

Compound Preparation

Compounds were dissolved in DMSO, then serially diluted in growth medium(RPMI 1640 medium supplemented with 100 U/ml penicillin G, 100 μg/mlstreptomycin, 10 mM L-glutamine) to give 4× the required concentrationrange in the presence of 0.04% DMSO. Each compound was assayed intriplicate at all concentrations.

Preparation of PBMCs

Defibrinated human blood from volunteers known to be allergic to Timothygrass was centrifuged at 2500 rpm for 15 minutes. The upper layer ofserum was collected and heat-inactivated at 56° C. for 30 minutes(HI-autologous serum). The lower layer of cells was resuspended in 50 mlPBS (+Ca+Mg), 25 ml diluted blood were overlaid onto 20 ml Lymphoprep in50 ml tubes then centrifuged at 2500 rpm for 20 minutes at RT. The bandat the serum/Lymphoprep interface was carefully removed. The collectedcells were washed with PBS and re-suspended at 4×106/ml in growth mediumwith HI-autologous serum. PBMCs were seeded at 0.4×106 cells/well inflat-bottomed 96 well plates in the presence of 10 ug/ml Timothy Grassantigen (Alk Abello) and test compounds at appropriate concentrations ina total volume of 200 ul.

Incubation and Cytokine Assays

Plates were incubated at 37° C. in 5% CO₂ for up to 6 days. The cellmedium from each well was harvested and stored at −20° C. prior toanalysis. Cytokines and chemokines in supernatants were detected usingMeso-Scale® Discovery 10 spot plates for Human TH1/Th2 cytokines.

In the above assay, data from separate studies with PBMCs from threeallergic donors showed Example 22 to reduce production of the Th2cytokines IL-5 and IL-13 in a dose response manner with ≥50% reductionobserved at 0.04M compared to the allergen control.

Examples 21 and 22 of the invention were also tested for in vivobiological activity in the following model:

Assay for the Induction of Interferon-α Following Intranasal Dosing inthe Mouse.

Compounds were dissolved in 0.2% Tween™ 80 in saline and administeredintranasally (5.1l in total between the nostrils) to female BALB/c mice(n=6) under general anaesthesia. Animals were euthanased 2 hours afterdosing and a terminal blood sample was taken and serum levels ofInterferon-α measured using an ELISA assay.

In this model Example 21 showed mean serum levels of Interferon-α of20326 μg/ml and Example 22 showed mean serum levels of Interferon-α of21029 μg/ml. No Interferon-α was detected in vehicle treated controls.

What is claimed is:
 1. A method of treating or preventing allergicdisease, inflammatory disease, or autoimmune disease in a human subjectin need thereof, the method comprising administering to the human anagent that is useful in the treatment or prevention of allergic disease,inflammatory disease, or autoimmune disease, and a compound of formula(I):

wherein: R¹ is (1S)-1-methylbutyloxy; m is an integer having a value of4 to 6; n is an integer having a value of 2 to 4; or a salt thereof. 2.The method according to claim 1 wherein the compound is selected fromthe group consisting of: 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[4-(1-piperidinyl)butyl]-7,9-dihydro-8H-purin-8-one;6-amino-9-[4-(hexahydro-1H-azepin-1-yl)butyl]-2-{[(1S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one;6-amino-9-[5-(hexahydro-1H-azepin-1-yl)pentyl]-2-{[(1S)-1-methylbutyl]oxy}-7,9-dihydro-8H-purin-8-one;and 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one.3. The method according to claim 1 wherein the salt is apharmaceutically acceptable salt.
 4. The method according to claim 1wherein the compound is a free base.
 5. The method according to claim 1wherein the agent that is useful in the treatment or prevention ofallergic disease, inflammatory disease, or autoimmune disease is antigenimmunotherapy, an anti-histamine, a steroid, an NSAID, a bronchodilator,methotrexate, a leukotriene modulator, monoclonal antibody therapy,receptor therapy, or an antigen non-specific immunotherapy.
 6. Themethod according to claim 1 wherein the allergic disease, inflammatorydisease, or autoimmune disease is asthma or rhinitis.
 7. A method oftreating or preventing allergic disease, inflammatory disease, orautoimmune disease in a human subject in need thereof, the methodcomprising administering to the human an agent that is useful in thetreatment or prevention of allergic disease, inflammatory disease, orautoimmune disease, and 6-amino-2-{[(1S)-1-methylbutyl]oxy}-9-[5-(1-piperidinyl)pentyl]-7,9-dihydro-8H-purin-8-one,or a salt thereof.
 8. The method according to claim 7 wherein the saltis a pharmaceutically acceptable salt.
 9. The method according to claim7 wherein the compound is a free base.
 10. The method according to claim7 wherein the agent that is useful in the treatment or prevention ofallergic disease, inflammatory disease, or autoimmune disease is antigenimmunotherapy, an anti-histamine, a steroid, an NSAID, a bronchodilator,methotrexate, a leukotriene modulator, monoclonal antibody therapy,receptor therapy, or an antigen non-specific immunotherapy.
 11. Themethod according to claim 7 wherein the allergic disease, inflammatorydisease, or autoimmune disease is asthma or rhinitis.